Accepted Manuscript

New advances in scanning microscopy and its application to study parasitic protozoa

Wanderley de Souza, Marcia Attias

PII: S0014-4894(17)30582-9
DOI: 10.1016/j.exppara.2018.04.018
Reference: YEXPR 7560

To appear in: Experimental Parasitology

Received Date: 4 November 2017

Revised Date: 10 April 2018
Accepted Date: 23 April 2018

Please cite this article as: de Souza, W., Attias, M., New advances in scanning microscopy
and its application to study parasitic protozoa, Experimental Parasitology (2018), doi: 10.1016/
j.exppara.2018.04.018.

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 ACCEPTED MANUSCRIPT
1 FOR EXPERIMENTAL PARASITOLOGY
2 REVIEW
3

4 New advances in scanning microscopy and its application to

5 study parasitic protozoa

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6

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9 Wanderley de Souza* and Marcia Attias

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11

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12 Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de
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13 Biofísica Carlos Chagas Filho, Instituto Nacional de Ciência e
14 Tecnologia em Biologia Estrutural e Bioimagens, and Centro
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15 Nacional de Biologia Estrutural e Bioimagens-CENABIO,

16 Universidade Federal do Rio de Janeiro, CCS-Bloco G, 21941-
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17 900, Rio de Janeiro, Brasil

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19 *Corresponding author. Email address: wsouza@biof.ufrj.br

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1 Abstract
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3 Scanning electron microscopy has been used to observe and

4 study parasitic protozoa for at least 40 years. However, field
5 emission electron sources, as well as improvements in lenses and
6 detectors, brought the resolution power of scanning electron

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7 microscopes (SEM) to a new level. Parallel to the refinement of
8 instruments, protocols for preservation of the ultrastructure,

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9 immunolabeling, exposure of cytoskeleton and inner structures of

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10 parasites and host cells were developed. This review is focused on
11 protozoan parasites of medical and veterinary relevance, e.g.,

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12 Toxoplasma gondii, Tritrichomonas foetus, Giardia intestinalis, and
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13 Trypanosoma cruzi, compilating the main achievements in
14 describing the fine ultrastructure of their surface, cytoskeleton and
interaction with host cells. Two new resources, namely, Helium Ion
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16 Microscopy (HIM) and Slice and View, using either Focused Ion
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17 Beam (FIB) abrasion or Microtome Serial Sectioning (MSS) within

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18 the microscope chamber, combined to backscattered electron

19 imaging of fixed (chemically or by quick freezing followed by freeze
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20 substitution and resin embedded samples is bringing an exponential

21 amount of valuable information. In HIM there is no need of
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22 conductive coating and the depth of field is much higher than in any
field emission SEM. As for FIB- and MSS-SEM, high resolution 3-D
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24 models of areas and volumes larger than any other technique allows
25 can be obtained. The main results achieved with all these
26 technological tools and some protocols for sample preparation are
27 included in this review. In addition, we included some results
28 obtained with environmental/low vacuum scanning microscopy and

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1 cryo-scanning electron microscopy, both promising, but not yet
2 largely employed SEM modalities.
3

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6 Key words: Cytoskeleton; FIB-SEM; Environmental scanning
7 electron microscopy; Helium ion microscopy; High resolution

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8 scanning electron microscopy; Parasitic protozoa

Abbreviations

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9

10 CFE - Cold field emission

11 Cryo-FE-SEM- cryo-Field Emission-SEM
12 EFF - endoflagellar form
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13 ER- endoplasmic reticulum
14 ESEM- Environmental Scanning Electron Microscope
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15 FIB- Focused ion beam

GDD - gaseous detector device
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17 HIM- Helion Ion Microscopy

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18 HR-SEM - High-resolution Scanning Electron Microscope

19 IVN- intravacuolar network
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20 LaB6 - Lanthanum hexaboride

21 MSS- microtome serial section
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22 PLA- pressure-limiting aperture

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23 PV- parasitophorous vacuole

24 PVM- parasitophorous vacuole membrane
25 SEM- Scanning Electron Microscope
26 TEM - Transmission electron microscopy
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1 Introduction
2 Although Max Knoll obtained some images in 1935, Scanning
3 Electron Microscopy (SEM) is considered to have started with
4 Manfred von Ardenne in 1937. Subsequently, other groups (see
5 Zworykin et al.,1942a) used the same principle (review in Wells and
6 Joy, 2006). Charles Oatley and co-workers further developed the

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7 equipment, which was then commercialized by Cambridge Scientific
8 Equipment in 1965. At that time, the resolution achieved was in the

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9 range of 50 nm (reviewed in Bogner et al., 2007).

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10 For many years, scanning electron microscopy (SEM) has been
11 used in the biomedical sciences, primarily to characterize the

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12 surfaces of organs, tissues, and cells, providing important
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13 information about the three-dimensional organization of such
14 surfaces. Improvements in SEM resolution have resulted from the
introduction of lanthanum hexaboride filaments and the field
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16 emission gun (Broers, 1975; Goldstein et al., 2003; Joy and Pawley,
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17 1992). In addition, the development of colloidal gold particles, which

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18 can be recognized by both secondary and backscattered electrons

19 (Erlandsen et al., 1990; Faulk and Taylor, 1971), allowed the use of
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20 SEM to detect and localize specific surface-exposed molecules. In

21 short, the advancements in SEM information result from innovations
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22 introduced both in the microscope itself and in methods of sample

preparation.
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24 It is not the purpose of this review to analyze the most basic

25 principles involved in the generation of electron beams, their
26 behavior in magnetic fields, their interaction with the sample to
27 generate secondary and backscattered electrons, the development
28 of electron detectors, etc. Excellent books (Bozzola and Russell,
29 1998; Goldstein et al., 2003) and reviews (Bogner et al., 2007;

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1 Hawkes, 2015) address these fundamental topics. However, it is
2 interesting to point out some basic information, relevant for better
3 understanding the process of obtaining high resolution with a
4 scanning electron microscope. First, it is crucial that the diameter
5 of the electron beam that will scan the sample surface is as small

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6 as possible. Second, it is necessary to generate a signal from the
7 specimen surface with an electron beam of variable energy (which

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8 depends on the voltage used to accelerate the electrons) so that
9 secondary electrons can come from different layers of the sample.

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10 The true surface can be observed using electrons of low energy,
11 with an accelerating voltage of approximately 1 kV. Third, the

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12 electrical conductivity of the probed surface must be perfect, to
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13 prevent any accumulation of electrostatic charge. In most
14 biological samples, the surfaces are not good conductors, so it is
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15 necessary to add a conductive coat to the sample. A metal coat 1

16 to 10 nm thick is considered to be thin. High-vacuum evaporation
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17 with noble metal alloys, such as gold-palladium, are ideal for

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18 obtaining images from secondary electrons. Iridium, tungsten or

19 platinum, although not as good as noble metals, are alternatives
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20 to consider, especially for backscattered electron imaging. It is

21 relevant to consider that coating with heavy metals increases the
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22 signal/noise ratio for samples of low atomic number. Thicker coat

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23 layers, however, may obliterate small surface structures, making

24 them invisible through the microscope. Non-conducting
25 specimens can be imaged, although at a lower resolution, without
26 coating, by using an environmental SEM (ESEM) or a low-voltage
27 mode of SEM operation, as will be discussed below. Fourth, it is
28 mandatory to have highly sensitive secondary and backscattered

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1 electron detectors that are well positioned in relation to the sample.
2 A quick look at papers that used SEM to analyze different cell
3 types shows clearly that this technique has been used to analyze
4 the surface topology of cells, tissues and even organs. In addition to
5 biological applications, SEM has been widely used in materials

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6 science to analyze all types of natural and artificial surfaces created
7 by processes such as fracture, abrasion, etc. (review in Wells and

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8 Joy, 2006). Applying these concepts to biological systems, we can
9 use SEM to visualize the true surface of the cells, as well as inner

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10 structures, including the cytoplasm, the nucleus, organelles and
11 cytoskeletal elements. The inner portions of the organelles can also

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12 be exposed using different approaches, as will be discussed below.
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13 With improvements in resolution, researchers have realized that
14 detailed information about the inner structural organization of cells
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15 could be obtained via SEM, provided that those structures could be

16 exposed. Soon, several protocols emerged, combining pre-existing
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17 and new approaches. Indeed, it became possible to expose the

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18 inner portions of cells using at least three approaches: cleaving,

19 freeze-fracture, and the use of detergents.
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20 The first views of the inner structures of cells by SEM were

21 obtained by cleaving compact tissue samples and exposing the
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22 cleaved surfaces to prolonged and successive incubations in the

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23 presence of osmium tetroxide (the so-called maceration process),

24 with subsequent critical-point drying. Examples of great success
25 with this approach come from the pioneering work of Tanaka and
26 colleagues (Tanaka, 1981), who obtained superb 3-dimensional
27 SEM images that revealed the architecture and distribution of
28 mitochondria, the endoplasmic reticulum, and the Golgi complex in
29 several cell types. When cells grow in a monolayer, as with several

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1 cell lines, cleavage becomes more complicated, because even if
2 they are scraped and compacted by centrifugation, the resulting
3 pellet is not usually firm enough to withstand the cleavage and
4 maceration processes. As a result, the cells tend to disassemble.
5 However, an approach for free cells or cells that grow in

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6 suspension was developed by Fukudome and Tanaka (1986). In
7 this method, the cells were embedded in gelatin or chitosan. Once

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8 jellified, this preparation was cut into small pieces and treated as
9 small tissue fragments. Although this method was proven to

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10 preserve membrane-bound organelles well, the natural orientation
11 of the cells was lost, and the cytoskeleton was not well preserved.

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12 The preservation of the cytoskeleton was achieved in a later work
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13 by the same authors (Fukudome and Tanaka, 1992). A second
14 approach is the exposure of the inner portion of the cells using the
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15 freeze-fracture technique (Moor et al., 1961). One critical step in

16 this technique is the process of cell freezing, which must be
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17 performed under conditions in which water crystals are not

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18 formed. This process can be achieved through the use of various

19 techniques, such as impact freezing (Heuser et al., 1979; Heuser
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20 and Kirschner, 1980) and high-pressure freezing (Moor et al.,

21 1980). After successful freezing, the samples can be fractured
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22 and etched, exposing the inner structures of cells. Much like the
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23 deep-etching technique that was developed by Heuser and

24 Kirschner (1980), the samples can then be imaged in the frozen
25 state using a cryo-scanning electron microscope (Walther, 2003).
26 These two approaches are best suited for observing membrane-
27 limited structures. We can also start sample preparation using
28 cryofixation followed by freeze-substitution and embedding in
29 epoxy resins which allow subsequent visualization of the cell

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1 structures. The observation of the cytoskeleton requires specific
2 approaches that will be covered in the technical appendix of this
3 review. Freeze-fracture methods have been used with great success
4 in several cell types to expose the cytoskeleton (Heuser and
5 Kirschner, 1980), and the final replica was observed by transmission

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6 electron microscopy (TEM). A third approach is the use of
7 detergents that partially or completely remove the plasma

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8 membrane and the soluble contents of the cytoplasm, allowing the
9 observation of the cytoskeleton using SEM. For each cell type,

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10 adaptations of the detergent mixture, time, and temperature used for
11 extraction are necessary (Lindroth et al., 1992; Svitkina et al., 1995).

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12 Most, if not all, of the methods described above have been
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13 developed and applied primarily to tissues or mammalian cells in
14 culture. However, protozoan parasites have some peculiarities. In
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15 particular, protozoan parasites are single cells, and many of them

16 spend part of their life cycles inside mammalian cells. More recently,
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17 we have adapted and developed several protocols to expose the

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18 inner structures of protozoan parasites, both isolated and inside their

19 host cells. These approaches provided new information about the
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20 organization of the cytoskeleton and certain organelles, as well as

21 new information about the organization of the parasitophorous
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22 vacuole, where some protozoa survive and multiply within

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23 mammalian host cells (Benchimol et al., 2004; de Andrade Rosa et

24 al., 2015, 2013; De Souza and Attias, 2015; Magno et al., 2005a;
25 Maia-Brigagão et al., 2013; Sant’Anna et al., 2005; Vatarunakamura
26 et al., 2005).
27 In this review, we will focus on the application of some
28 approaches to prepare biological samples, using parasitic protozoa
29 as examples. We would like to point out that these approaches have

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1 been underused to analyze the structure of parasitic protozoa.
2 That is the primary reason for the fact that most of the illustration
3 will come from papers published by our group. As a consequence,
4 this review may serve as inspiration for the development of the
5 research of others. Next, we will analyze four major topics

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6 involving these methodologies (Environmental SEM, High-
7 Resolution SEM, Helion Ion Microscopy (HIM), and Cryo-SEM

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8 and we will explore the potential to achieve three-dimensional
9 reconstruction of biological structures using surfaces exposed

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10 either by a focused ion beam (FIB) (Heymann et al., 2006) or by
11 the sequential removal of the top surface of the sample using a

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12 diamond knife at room temperature or at very low temperatures
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13 using the same principles used for cryosectioning (Tokuyasu,
14 1973). In addition, we include as supplemental information some
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15 sample preparation methods for high-resolution scanning

16 microscopy imaging.
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17
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18 1. Low-Vacuum/Environmental SEM. Principles and

19 applications.
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20 The main paradigm broken by low-vacuum or environmental

21 SEMs (ESEM) is that the microscope column and the specimen
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22 chamber should be kept under a vacuum of at least 10-5 Torr so

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23 that the electron beam that originates at the source can reach
24 the sample without being scattered by gas molecules (Goldstein
25 et al., 2003). In these instruments, which are more appropriately
26 called variable-pressure SEMs, the specimen chamber vacuum
27 can operate in a range from 10-5 Torr (high vacuum) to 0.1 to 20
28 Torr. In this last instance, the chamber can carry hydrated

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1 samples, such as a liquid droplet containing hydrated samples. In
2 ESEM instruments, the specimen is placed in a relatively high-
3 pressure chamber, and the electron optical column is differentially
4 pumped to keep the vacuum adequately low at the electron gun.
5 The high-pressure region around the sample in the ESEM

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6 neutralizes the charge of the sample and amplifies the secondary
7 electron signal. The ESEM operates with an electron beam that

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8 can be generated from either tungsten or LaB6 filaments or a field
9 emission gun, with superior resolution due to the high brightness of

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10 the primary electrons and their small spot size, even at low
11 accelerating potentials. To prevent charging of non-conductive

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12 specimens, the operating conditions must be adjusted so that the
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13 incoming beam current is equal to the sum of the outgoing
14 secondary and backscattered electron currents, a condition that is
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15 more often met at accelerating voltages between 0.3 and 4 kV.

16 Between the column and the sample chamber, the introduction of a
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17 pressure-limiting aperture (PLA) of variable diameter restrains the

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18 flow of gas between the chamber and the column. Once the
19 electron beam reaches the chamber, some of the electrons are
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20 scattered by the gaseous molecules in the chamber. Other

21 problems to be circumvented require the correct adjustment of
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22 parameters such as the acceleration voltage, the spot size of the

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23 beam and grounding, which must be defined for each sample. The
24 secondary electrons generated are captured by a gaseous detector
25 device (GDD) that utilizes the gas molecules in the chamber to
26 amplify the signal. However, to detect backscattered electrons, the
27 same detector used for high-vacuum SEM is compatible. A last
28 obstacle to overcome is that the exposure of a hydrated sample to
29 the low-vacuum conditions of the ESEM chamber does not avoid

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1 progressive dehydration and beam damage to the sample.
2 These effects are less harmful for samples that are naturally
3 hard, such as the cell wall of plants, the cuticle and exoskeleton
4 of worms and insects, and minerals. For protists, which
5 necessarily live in an aqueous environment, the parameters of

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6 the ESEM chamber are quite harsh. Nevertheless, the potential
7 of this approach was shown by Maia-Brigagão and de Souza

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8 (2012) in analyzing the adhesion of Giardia lamblia to
9 mammalian cells in culture. Using this approach, the authors

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10 could quantify the parameters of parasite adhesion to the cells,
11 avoiding the detachment of the parasites from the substrate due

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12 to processing procedures. The parasites were allowed to interact
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13 with Caco-2 cells, mounted directly (without subsequent post-
14 fixation, dehydration, critical-point drying and metal coating) on a
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15 proper stub and immediately observed under low-vacuum

16 conditions (50 Pa).
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17 Environmental microscopy is potentially very useful for

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18 analyzing basic aspects of parasite-host cell interactions.

19 However, to date, this technique has not been widely used,
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20 probably because few laboratories are equipped with such

21 equipment despite its relatively low cost. However, we may
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22 expect an expansion on its use in the next few years since it is

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23 gradually becoming available in several EM facilities. Besides

24 that, sample preparation is relatively fast. As an example,
25 observations of the surface of the nematode parasite Trichuris
26 muris and its interaction with the host tissue using Low Vacuum
27 SEM and Environmental SEM using freshly fixed or unfixed
28 hydrated samples preserved the secretory products of the
29 bacillary glands on the surface of the parasite, and the mucous

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1 material and eggs on the intestinal surface (Lopes Torres et al.,
2 2013). In this sense, one suggestion would be the observation of
3 parasites in situ, e.g. Toxoplasma gondii oocysts in the feline ileum,
4 or the comparison of mucous secretion in epithelia infected and
5 non-infected by Entamoeba, Giardia or Trichomonas. However, the

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6 low resolution of the method, and the rapid dehydration of the
7 sample, when compared to high vacuum SEM, are drawbacks not

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8 yet overcome.
9 2. High-resolution SEM (HR-SEM) and Helion Ion Scanning

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10 Microscopy (HIM).
11 In SEM, resolution can be defined as the minimal area of the

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12 electron beam that can generate a detectable signal from the
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13 surface of the specimen. However, the interaction volume that
14 results from electron beam scattering enhances this area, lowering
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15 the resolution power of SEM. For several years, SEM resolution

16 was limited by the fact that the virtual source size that a thermionic
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17 tungsten filament can reach at 20 kV is between 30 and 100 µm;

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18 furthermore, under these conditions, the volume of interaction is

19 increased by the deeper penetration of the beam into the sample,
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20 lowering the actual resolution power (Goldstein et al., 2003). This

21 shortcoming was surpassed first by LaB6 filaments, which provide a
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22 brightness 5-10 times greater than that achieved with tungsten

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23 filaments and a source size that ranges from 5 to 50 µm.

24 Subsequently, cold and thermal field emission guns reached a
25 probe size of less than 5 nm. With the in-lens system, a probe with
26 a diameter of 1.2-1.8 nm and a current of 1 pA at 30 kV was
27 achieved (Goldstein et al., 2003; Joy and Pawley, 1992). Combined
28 with improvements in lenses and detectors (both for secondary and
29 backscattered electrons), a plethora of new possibilities arose for

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1 studying biological samples at the subcellular level. Some of
2 these microscopes allow the observation of large samples. In
3 other microscopes, the sample is positioned inside a gap of the
4 split objective lens pole piece, a configuration known as in-lens;
5 such samples are necessarily smaller. A new generation of high-

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6 resolution SEMs, which are currently available from most
7 commercial companies, work with beams with a diameter as

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8 small as 0.4 nm, thus providing resolutions that range from 0.4
9 (at 30 kV) to 1.2 nm (at 1 kV). Only data obtained with such

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10 equipments will be emphasized in this review. Cold field
11 emission (CFE) guns made it possible to magnify high-resolution

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12 images up to 2 million times.
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13 Another novel scanning microscopy technology that emerged
14 recently is HIM, which uses helium ions rather than electrons
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15 (Economou et al., 2012). In HIM, the ion emission originates

16 preferentially from only three atoms at the apex of the source, and
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17 only one ion is selected for imaging. The extracted ions are
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18 accelerated down the column of the microscope much in the same

19 fashion as in SEM, but because the gas field ionization source
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20 has an ultra-high brightness, a very small beam defining aperture

21 may be used, avoiding spherical and chromatic aberrations and
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22 resulting in a greater depth of field and an estimated resolution of

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23 0.35 nm (Chen et al., 2011; Joens et al., 2013). The secondary

24 electron yield is much higher than that obtained with electron
25 microscopy, resulting in a better signal-to-noise ratio and thus
26 better images (Bell et al., 2009; Inai et al., 2007; Joens et al.,
27 2013). In addition, the He beam does not induce charging,
28 avoiding the need for conductive coating of the samples and thus
29 improving significantly the spatial resolution of the images. The

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1 smaller beam, and the narrower angle of convergence in
2 comparison to an electron beam, also allows the acquisition of
3 images with a larger depth of field (Bell et al., 2009). This
4 combination of characteristics results in the ultra-high resolution
5 microscope, which is a superb tool for analyzing true biological

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6 surfaces, as shown by Rice et al. (2013), who characterized the
7 surface of kidney cells, and by Vanden Berg-Foels et al. (2012), who

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8 analyzed bone tissue. HIM was also used to observe colon cancer
9 cells (Bazou et al. 2011), plants, HeLa cells, bacteria and

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10 nematodes (Joens et al. 2013) and by Păunescu et al. (2014), who
11 described new aspects of the male reproductive tract of rats. After

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12 this short description of the state of the art of high-resolution
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13 scanning microscopy, we present some examples of its application
14 to analyze structures of pathogenic protozoa, such as (a) the actual
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15 surface of the cell, (b) the flagellum, (c) special structures of the
16 cytoskeleton, and (d) the interaction of intracellular protozoa with
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17 host cells.
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18 2.1. The cell surface

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19 The general concept that the cell surface of protists is smooth, as

20 suggested by the heavier conductive coating necessary in
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21 conventional SEM, has changed drastically with thinner coating of

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22 specimens with chromium or carbon for high-resolution scanning

23 electron microscopy (HR-SEM). Rougher surfaces and the presence
24 of small invaginations, clefts, secretion vesicles and specialized
25 areas became apparent. Differences in cap formation between the
26 invasive Entamoeba histolytica and the non-invasive E. dispar were
27 described by Chávez-Munguía et al. (2012). Using concanavalin-A
28 as a tracer, those authors showed that while on the E. hystolytica

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1 surface, the tracer is quickly discarded in small vesicles
2 concentrated at the posterior end of the cell (uroid region), in E.
3 dispar, small patches of vesicles were distributed randomly.
4 Before that time, HR-SEM had also been used by Aguilar-Díaz et
5 al. (2010) to show the filamentous structure of cyst-like structures

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6 (CLS) induced by Entamoeba trophozoites in vitro. Similarly, the
7 initial secretion of cyst wall components in trophozoites of Giardia

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8 lamblia was demonstrated by Erlandsen et al. (1990), as was the
9 filamentous structure of the cyst wall, combining structural

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10 evidence with immunolabelling of cyst wall components (Figure 1).
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12 HR-SEM also revealed the diversity in the area and topology of
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13 the membrane between the cytostomal entrance and the flagellar
14 pocket of epimastigotes of Trypanosoma cruzi. These features
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15 were initially shown by TEM examination of freeze-fracture

16 replicas (Martinez-Palomo et al. 1976; Pimenta et al. 1988) of T.
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17 cruzi epimastigotes and later by Nakamura et al. (2005) using HR-

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18 SEM. This area shows a concentration of receptors for the lectin

19 concanavalin-A, as well as a rougher texture in relation to the rest
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20 of membrane surface (Supplemental Figure 1).

21
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22 More recently, using HIM, which provides a resolution of 3.5 nm

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23 in uncoated samples, it was possible to observe even finer details

24 of the cell surface. One good example is the surface of
25 extracellular Toxoplasma gondii tachyzoites showing that the
26 contours of the polar ring and the conoidal fibers that rise above
27 (Figure 2A) in the apical portion of the cell (de Souza and Attias,
28 2015). The cell body of T. gondii presented striations along its
29 length, compatible with the pattern of distribution of the

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1 subpellicular microtubules (Figure 2A, B), except in the more apical
2 portion of the cell. Tubular structures of an unknown nature were
3 observed in the apical portion of extracellular tachyzoites (Figure
4 2B). These structures could be remnants of the PV membrane or of
5 some membranous structure of the host cell from which this parasite

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6 was liberated. The tubular structures were also compatible in
7 diameter with filopodia and microvilli of the host cell, to which

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8 parasites often adhere (A MacLaren et al., 2004). It is worthy to
9 mention that the results in MacLaren’s paper were obtained with a

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10 cold field emission gun. Another difference between the surface of
11 intracellular and extracellular parasites is that soon after invasion, at

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12 the stage of a single parasite per cell, intracellular parasites
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13 presented many structures of variable size and shape, suggestive of
14 intense secretion (Figure 2C). Those structures were scattered all
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15 over the cell body and varied in size and in shape, from tubular to
16 hemispherical. These membranous assemblies were not seen at the
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17 apical extremity.
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18 2.2. Visualization of cytoskeletal structures

19 The observation of the cytoskeleton by either HR-SEM or HIM
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20 requires a different approach. Initially, freeze-fracture methods,

21 including a deep-etching step, were used with great success to
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22 expose the cytoskeleton in several mammalian cell types, with the

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23 final replica observed by TEM (Heuser and Kirschner, 1980).

24 These methods were also used to visualize protozoa, such as
25 trypanosomatids (Souto-Padrón et al., 1984), Tritrichomonas foetus
26 (Benchimol et al., 1993), Giardia intestinalis (Kattenbach et al.,
27 1996), and Toxoplasma gondii (Lemgruber et al., 2010). Another
28 approach developed that was proved to be very effective for
29 observing elements of the cytoskeleton by HR-SEM, avoiding the

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1 steps of freezing, fracturing and replica generation, which require
2 specific equipment, was the lysis and removal of the plasma
3 membrane and the soluble contents of the cytoplasm using
4 detergents. Combinations of detergents have been applied to
5 potential host cells, such as macrophages (del Caro et al., 1989),

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6 and several protozoans, including non-pathogenic kinetoplastids,
7 such as Bodo sp. (Attias et al., 1996), Giardia (Holberton and

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8 Ward, 1981), Trypanosoma (Sant’Anna et al., 2008) and
9 Tritrichomonas foetus (Benchimol 2005; de Andrade Rosa et al.,

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10 2013). All these protocols follow the same rationale of detergent
11 extraction, followed by chemical fixation, dehydration, critical-

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12 point drying, conductive sputtering and observation via HR-SEM.
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13 Some of these approaches are described in detail in the
14 technical appendix. Most protocols use a combination of nonionic
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15 detergents (Triton-X100, NP-40), HEPES and PIPES buffers,

16 and, sometimes, drugs that stabilize microtubules. The time and
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17 temperature of incubation are highly variable, as is the index of

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18 ideally extracted cells. In conclusion, for each cell type, several

19 parameters, besides the choice of detergent itself (Lindroth et al.,
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20 1992), must be established. These parameters include the

21 concentrations of detergents, buffers, the time of exposure,
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22 temperature, etc. However, this procedure may remove proteins

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23 associated to the main cytoskeletal structures and this possibility

24 must be taken in consideration in the interpretation of the results
25 obtained.
26 HR-SEM and HIM were recently used to analyze in more detail
27 the cytoskeletal structures of Tritrichomonas foetus (de Andrade
28 Rosa et al., 2015) and Giardia intestinalis (Gadelha et al., 2015),
29 as will be detailed below.

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1 2.2.1. The cytoskeleton of Tritrichomonas foetus
2 The Trichomonadidae family comprises species such as
3 Trichomonas vaginalis and T. foetus, which are agents of serious
4 human and animal infections, respectively. In addition, these
5 species are also good models for studying early eukaryotic cells,

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6 since in addition to the organelles that are found in all eukaryotic
7 cells, these organisms contain other organelles, such as

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8 hydrogenosomes and a complex and elaborate cytoskeleton, which
9 constitutes the mastigont system (Benchimol, 2004; Honigberg et

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10 al., 1971). This system includes the following structures: (a) the
11 pelta-axostylar complex, which is a well-organized array of

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12 microtubules that constitutes an axial skeleton with stable,
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13 longitudinally oriented microtubules that extend along the length of
14 the cell, (b) four flagella, three anterior and one recurrent, which are
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15 all associated with basal bodies, (c) a large striated root fibril that is
16 found only in trichomonads that have an undulating membrane,
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17 known as a costa, and is thought to provide mechanical support for

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18 this structure, and (d) other structures, such as the parabasal

19 filaments, which are thin striated root fibrils that connect the basal
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20 bodies to the first Golgi cisternae, possibly to support this organelle

21 (Benchimol et al., 1993; Consort Ribeiro et al., 2001). In addition,
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22 the filaments and lamellae that are associated with the basal bodies
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23 are not well characterized (Viscogliosi and Brugerolle, 1994).

24 The structure of the cytoskeleton of trichomonads has been
25 analyzed using several techniques (e.g., immunofluorescence
26 microscopy (Batista et al., 1988; Benchimol and De Souza, 1987;
27 Viscogliosi and Brugerolle, 1993), high-voltage TEM of whole cells
28 (Benchimol and De Souza, 1987) and TEM of thin sections (Batista
29 et al., 1988; Benchimol and De Souza, 1987; Granger et al., 2000;

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1 Ribeiro et al., 2000), negative staining, freeze-fracture and
2 freeze-etching (Benchimol et al., 2000, 1993; Kattenbach et al.,
3 1996). Here, we will emphasize only those structures for which
4 HR-SEM provided new information.
5 2.2.1.1. Pelta-axostylar system

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6 The pelta-axostylar system is readily seen by conventional
7 SEM after plasma membrane removal. In most cases, this

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8 system is visualized as very smooth or with discrete grooves
9 (Benchimol, 2005). When observed with HR-SEM, the axostyle

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10 (Figure 3) appears as a hollow tube formed by a sheet of
11 microtubules, which open in the anterior region of the cell and

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12 form a spoon-like area known as a capitulum. The pelta was
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13 visualized as a crescent-shaped ribbon of microtubules on the
14 internal face of the capitulum, which extends to apically surround
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15 the walls of the periflagellar canal. Furthermore, the extremities

16 of the pelta microtubules were seen for the first time with HR-
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17 SEM and could be easily distinguished from the axostyle

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18 microtubules, as these sets of microtubules form two assemblies

19 (Figure 3) with different orientations. The pelta-axostylar junction
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20 was clearly observed for the first time using HR-SEM (Figure 3).
21 Earlier observations had shown that axostyles were formed by
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22 adjacent microtubules connected by filamentous bridges

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23 (Benchimol et al., 2000, 1993; Mooseker and Tilney, 1973). These

24 bridges were observed with HR-SEM, appearing as 10.4 ± 1.0 nm
25 thick filaments spaced at intervals ranging from 9 to 25.4 nm
26 (Figure 3C).
27 2.2.1.2 Costa and accessory structure
28 HR-SEM showed that the costa presents an accessory
29 structure that runs along its right-hand side (Figure 4). This

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1 structure has a striated pattern but originates in sites with opposite
2 directions (Figure 4B). Previous analysis of replicas of quick-frozen,
3 freeze-fractured, deep-etched and rotary-replicated cells revealed
4 the presence of pits at the periphery of the costa (Benchimol et al.,
5 1993). TEM of thin sections also revealed the presence of a

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6 structure that may correspond to the costa accessory filament.
7 However, a perspective view of the structure was achieved only with

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8 HR-SEM (Figure 4 A and B).
9 A network of filaments that connect the costa to the undulating

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10 membrane and was previously seen only using freeze-fracture and
11 deep-etching (Benchimol et al., 1993) was visualized by HR-SEM

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12 (Figure 4C). This network is composed of 12.5 ± 1.2 nm thick
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13 filaments, which may reach a length of 73.6 ± 9.1 nm, and of
14 globular structures with a mean diameter of 29.20 ± 3.3 nm. The
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15 images also revealed that the costa interacted with other

16 cytoskeletal structures, such as the comb (Figure 4C and D), which
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17 connects the costa to the infrakinetosomal body and has been

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18 described as a scalariform structure that is found in T. foetus but not

19 in Trichomonas vaginalis (de Andrade Rosa et al., 2013; Viscogliosi
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20 and Brugerolle, 1993). The comb appeared to be formed of small

21 filaments that were oriented perpendicularly in relation to the costa
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22 (Figure 4).
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23 2.2.1.3 Other filaments

24 High-resolution SEM also allowed the clear observation of the
25 parabasal filaments, which are striated structures that have been
26 implicated in the support of the large Golgi complex of
27 trichomonads. These filaments appeared as striated fibers that are
28 thinner and shorter than the costa, although their periodicity (40.2 ±
29 5.4 nm) was rather similar. The parabasal filaments and costa were

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1 observed to be connected to a network of 12.2 ± 2.5 nm thick
2 cytoskeleton filaments, as well as to the nucleus.
3 High-resolution SEM also allowed the visualization of the
4 sigmoidal filaments, which are sigma-shaped striated roots that
5 were previously observed by conventional TEM in the anterior

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6 region of T. foetus (Viscogliosi and Brugerolle, 1993). These
7 filaments are formed by at least six filaments, which are located

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8 near the pelta and the axostyle (Figure 4D). HR-SEM allowed the
9 observation of striations with a periodicity of 50.5 ± 6.6 nm.

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10 It has been shown that under unfavorable environmental
11 conditions, such as abrupt changes in temperature (Granger et

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12 al., 2000; Pereira-Neves et al., 2012) or the presence of drugs,
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13 such as colchicine (Madeiro da Costa and Benchimol, 2004), the
14 trophozoite of T. foetus acquires a rounded or elliptical shape and
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15 internalizes its flagella, transforming into an endoflagellar form

16 (EFF), which is also known as a pseudocyst because a cyst wall
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17 is not formed (Pereira-Neves et al., 2003). Previous studies using

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18 light and conventional SEM and TEM have shown that

19 morphological changes occur during the transformation of
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20 trophozoites into pseudocysts (Meyer Mariante et al., 2004;

21 Pereira-Neves et al., 2003; Pereira-Neves and Benchimol, 2009).
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22 SEM analysis showed that the flagella were externalized and the
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23 axial axostyle presented a region where the microtubules turned

24 upon themselves. In addition, the axostyle presented a concave
25 region, where the nucleus is surrounded by a network of filaments
26 that are visible by HR-SEM (Supplemental Figure 2). A network of
27 12.5 ± 1.2 nm thick filaments was seen connecting the periphery
28 of the costa accessory filament to the region where the recurrent
29 flagellum attaches to the protozoan cell body, forming the

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1 undulating membrane. The nucleus was positioned in the center of
2 the axostyle, the pelta involved the periflagellar canal, and the costa
3 was curved, allowing this axial structure to follow the shape of the
4 rounded cell (Supplemental Figure 2). The axostyle was smaller and
5 had an irregular edge in the multinucleated pseudocyst, suggesting

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6 fragmentation of this structure. The costa also underwent
7 morphological modifications in the multinucleated pseudocysts,

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8 displaying a hooked or U shape (Supplemental Figure 2 B and C),
9 and was localized around the axostyle. The curved costa could

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10 present several shapes, but its size did not vary significantly.
11 Unexpectedly, the accessory filament and the filament network

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12 connecting the costa to the recurrent flagellum region were no
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13 longer observed in the pseudocyst cytoskeleton.
14 2.2.3 The cytoskeleton of Giardia intestinalis
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15 The Giardia cytoskeleton is characterized by the presence of

16 stable microtubular structures that include the adhesive disk, four
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17 pairs of flagella, the funis and the median body (Friend, 1966;
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18 Holberton, 1973; Sant’Anna et al., 2005). The adhesive disk is

19 crucial for the attachment of Giardia to the host intestinal surface
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20 (Campanati et al., 2002; Holberton, 1973; Woessner and Dawson,

21 2012). This structure is formed by a spiral layer of microtubules
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22 adjacent to the plasma membrane in the ventral region of the cell

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23 and is physically connected to the cell by small filaments (Holberton,

24 1973). Microribbons, which contain giardins, extend from the
25 microtubules towards the cytoplasm and are connected to each
26 other by cross-bridges (Holberton, 1973; Terra et al., 2010).
27 Giardia has four pairs of flagella: (1) anterior, (2) lateral-posterior,
28 (3) ventral and (4) caudal, which emerge from the basal bodies
29 localized in the anterior region of the cell (Crossley et al., 1986;

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1 Sant’Anna et al., 2005). The marginal plates, which have been
2 described as part of the ventrolateral flange, are also associated
3 with the axonemes of the anterior flagella (Friend, 1966; Maia-
4 Brigagão et al., 2013). The ventral flagella typically present a
5 membrane projection that is filled with a dense material (Friend,

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6 1966; Holberton, 1973). An array of microtubules called the funis
7 follows the internal portion of the axonemes of the caudal flagella

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8 (Benchimol et al., 2004; Carvalho and Monteiro-Leal, 2004).
9 Another microtubular element called the median body has been

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10 described (Feely et. al., 1990). HR-SEM of the cytoskeleton of G.
11 intestinalis (Figure 5 A) showed that the lateral crest presented

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12 small filaments that connected the crest to the ventral disk (Figure
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13 5B). The “bare area” contains banded collars that are associated
14 with the axonemes of the flagella, and the upper side of the
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15 marginal plates is also linked to a network of filaments (Figure

16 5C). To examine the trophozoite cytoskeleton, several trials were
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17 necessary using different extraction conditions, until a good

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18 preservation of structural elements was achieved. Using living

19 cells incubated with 2% NP-40 before fixation, several cytoskeletal
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20 elements were exposed. The adhesive disk, the ventrolateral

21 flange, the median body and the funis were preserved in this
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22 preparation. Although these structures were visualized previously,

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23 the combination of membrane extraction and analysis using HR-

24 SEM or HIM revealed novel details concerning the surface
25 characteristics of the structural elements, as described below.
26 Novel characteristics of the lateral crest, which presented small
27 filaments that had a different orientation than the disk
28 microtubules (Figure 5B, C) and were connected to the disk
29 microtubules, could be seen. Detergent extraction using 2% NP-

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1 40 plus 2% Triton X-100 before fixation exposed the cell’s interior,
2 allowing the observation of the area where the basal bodies, the
3 axonemes, and the banded collars are found (Figure 5C, D). In cells
4 adhered through their ventral regions, two types of banded collars
5 were seen and numbered as banded collar number 1 (BC1) and

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6 banded collar number 2 (BC2). Both of these types are located in
7 the anterior region of the cell, dorsally to the disk, but in opposite

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8 positions (Figure 5 C, D). BC2 appeared as a rope-shaped
9 structure, presenting horizontal segments connected by short

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10 bridges (Figure 5 C, D). The ventral disk microtubules emerged
11 from the basal bodies surrounded by BC2, following a spiral course.

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12 However, when Giardia was observed from a ventral view, the
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13 banded collars exhibited the opposite shape, and another set of
14 microtubules that also contacted the ventral disk could be observed.
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15 This set of microtubules emerged from the other BC2, which was
16 continuous with the left axoneme of the caudal and ventral flagella.
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17 In this position, another BC1 that joined the right axonemes of the
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18 caudal and ventral flagella was observed. The two sets of

19 microtubules and the BC2s associated with the respective basal
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20 bodies formed a goblet-shaped structure, as revealed by HIM.

21 Fibrilar projections connecting the axonemes were also observed in
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22 these preparations (Figure 5B).

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23 HR-SEM of extracted cells revealed structures associated with the

24 axonemes of the anterior flagella. The marginal plates were
25 observed in close contact with the axonemes of the anterior flagella
26 (Maia-Brigagão et al., 2013). In their upper portions, these plates
27 were in contact with a network of filaments (Figure 5B). This network
28 was observed continuously with a set of filaments, which extended
29 towards the periphery of the trophozoite and were parallel to the

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1 main cell axis. Trophozoites adhered through the ventral region
2 exhibited a network of filaments in the region corresponding to the
3 ventrolateral flange. These filaments were continuous with a
4 looser meshwork of filaments that spread out along the dorsal
5 region.

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6 Using HIM, cytoplasmic filaments were observed contacting
7 cytoskeletal structures, such as the median body. These filaments

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8 measured 9.4±1.2 nm in diameter. In some cases, ring-like
9 structures could be also seen.

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10 A detailed visualization of the funis, which is composed of
11 microtubular sheets associated with the axoneme of the caudal

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12 flagella, was also performed. HIM analysis revealed a lattice-like
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13 array of material covering the microtubular sheets of this structure.
14 The lateral microtubules emanated from the main structure and
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15 ran towards the axoneme of the posterior-lateral flagella , as

16 previously described (Benchimol et al., 2004). The funis
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17 microtubules were interconnected by bridges, which exhibited

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18 different lengths. In the final portion of the caudal complex, the

19 lateral microtubules were seen associated with cytoplasmic
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20 filaments.
21 The cytoplasmic and ventrolateral flange filaments were better
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22 preserved in this preparation. When Triton X-100 detergent was

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23 added to the extraction solution, the disk bridges were broken up,
24 as reported previously (Holberton and Ward, 1981). Despite that
25 finding, a more complete extraction of the Giardia membrane and
26 cytoplasm contents, which was achieved by combining both
27 detergents, allowed the detailed visualization of the kinetosomes
28 and their anchorages, revealing new information about the disk
29 origin and the virtually unknown behavior of the banded collar.

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1 Detergent extraction exposed an area known as the “bare area,”
2 where the axonemes, basal bodies and banded collars were
3 observed. Using HR-SEM and HIM, it was shown that two types of
4 banded collars are present in the cell (i.e., BC1 and BC2). These
5 collars were repeated on both sides of the cell. The BC2s present

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6 horizontal segments connected by small bridges that corresponded
7 to plaque, with light and dark lines, as reported previously (Crossley

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8 et al., 1986).
9 Feely, Erlandsen, Chase, Holberton (1990), and Erlandsen (1990)

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10 reported that the Giardia basal bodies were arranged in tetrads.
11 Each tetrad was associated with a banded collar and attached to the

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12 anterior pole of the nucleus. Using HR-SEM, it was shown that BC1
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13 wrapped the basal bodies of the right caudal/posterior-lateral flagella
14 when the cells were observed dorsally and the left caudal/ventral
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15 flagella when the cells were observed ventrally. The BC2s were
16 continuous with the basal bodies of the left caudal/posterior-lateral
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17 flagella (dorsal view) and the right caudal/ventral flagella (ventral

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18 view). Each BC2 presented a set of microtubules: the disk

19 microtubules emerged from the basal bodies associated with the left
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20 BC2, and the previously described supernumerary microtubules

21 emerged from the basal bodies associated with the right BC2. The
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22 consequence of the connection between the banded collar and

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23 these flagella axonemes is that these structures can migrate

24 together during mitosis. Feely, Erlandsen, Chase, Holberton, and
25 Erlandsen (1990) reported that isolated banded collars have the
26 capacity to nucleate new microtubules. Using electron tomography
27 (Schwartz et al., 2012), it was shown that microtubule-microribbon
28 complexes originated from dense bands in a region near the caudal
29 and posterior-lateral basal bodies. Thus, the banded collars alone or

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1 in combination with the basal bodies may represent microtubule
2 organizing centers that drive the formation of a new adhesive disk.
3 The sets of microtubules that emerge from BC2 form a single
4 goblet-shaped structure, suggesting that the spiral of the ventral
5 disc would be formed by the outer and inner (supernumerary

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6 microtubules) sets of fibers.
7 HR-SEM also revealed that the upper portion of the marginal

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8 plate is associated with a network of filaments that are continuous
9 with filaments parallel to the main cell axis, generating what has

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10 been designated as the ventrolateral flange. This structure was
11 described previously as a fibrous structure of paracrystalline

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12 regularity (Holberton, 1973). The adhesive role of the ventrolateral
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13 flange has been described previously (Erlandsen et al., 2004;
14 Feely et al., 1982), although the biochemical basis for this
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15 property remains unclear. On the dorsal side of the trophozoites,

16 filaments similar to those associated with the marginal plates were
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17 visualized, suggesting that these filaments comprise a single

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18 network. A meshwork of cytoplasmic filaments with a diameter of

19 9 nm was observed. In this region, labeling for actin was
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20 observed, suggesting that these filaments may correspond to

21 actin filaments, which could act as a scaffold and provide support
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22 for the dorsal surface and cellular components. These results

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23 corroborated previous immunofluorescence data, which showed

24 actin labeling throughout the entire trophozoite (Castillo-Romero
25 et al., 2009; Paredez et al., 2011). It has also been demonstrated
26 that the in vitro polymerization of Giardia actin produces filaments
27 (Paredez et al., 2011). The presence of cytoplasmic filaments with
28 a diameter of 12 nm was demonstrated in a previous study that
29 used replicas from deep-etched cells (Kattenbach et al., 1996).

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1 However, the presence of such filaments has not yet been
2 corroborated with alternative electron microscopy techniques.
3 HIM allowed the visualization of a material in a lattice-like array
4 that covered the microtubular sheets of the funis (Gadelha et al.,
5 2015) . In a previous work (Paredez et al., 2011), an actin helix was

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6 demonstrated to bundle the axonemes of the caudal flagella.
7 Bridges of different lengths interconnected the lateral microtubules

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8 of the funis, suggesting that these bridges were not stiff elements.

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9

10 2.3. Interaction of protozoa with host cells

11
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The interaction process of parasitic protozoa involves the
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12 adhesion of the parasite to the host cell, which induces a response
13 from the host cell. Therefore, the surfaces of the two cells play a
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14 fundamental role in this process. This process has been analyzed to

some extent using different microscopy techniques. SEM is a
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15

16 suitable method, since it allows the observation of the two

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17 interacting cells in some detail. However, to date, few studies on this

18 subject have been performed using high-resolution scanning
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19 techniques. Here, we will review basic aspects of the interactions of

20 Trypanosoma cruzi and Toxoplasma gondii with host cells.
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21 2.3.1. Trypanosoma cruzi

22 Several studies have shown that both the trypomastigote and

23 amastigote forms of T. cruzi are able to enter host cells and develop
24 an intracellular cycle. The process of “entrance” is a typical
25 endocytic process that involves the formation of a parasitophorous
26 vacuole. Several studies have shown that several surface-exposed
27 parasite proteins are involved in this process. In addition, several
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1 components of the host cell membrane (including the cell coat and
2 membrane-associated cytoskeletal components), such as lipid
3 rafts and several receptors, are involved in the participation of
4 dynamin, caveolin, and flotilin in the endocytic process. In
5 addition, macropinocytosis and phagocytosis take place during

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6 this interaction (reviews in Barrias et al., 2013; de Souza et al.,
7 2010). Images obtained from the interaction of amastigotes with

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8 host cells showed that amastigotes initially attach to microvilli,
9 followed by the formation of plasma membrane extensions that

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10 resemble a cup-like structure and extend upwards around the
11 parasite (see Fig. 1 in Fernandes et al., 2013). The exposure of

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12 the host cell cytoplasm via detergent extraction revealed a
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13 network of 5-7 nm thick filaments surrounding the amastigotes.
14 Figure 6 A, B, C and D show examples of the interaction
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15 processes of trypomastigotes and amastigotes with macrophages.

16 Surface projections and microvesicles, including exosomes, can
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17 be seen. The attachment of the parasite to the host cell surface

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18 triggers the formation of pseudopods, which surround the

19 parasite, leading to its subsequent internalization. This process
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20 can be interrupted by compounds that interfere with dynamin, a

21 GTPase that is involved in membrane fusion, as shown in Figure
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22 6D. Certainly, HR-SEM has great potential for adding new and
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23 relevant information, especially information about the contribution

24 of intracellular structures of the host cell to the formation of the
25 PV.

26 2.3.2. Toxoplasma gondii

27 HR-SEM was used to analyze basic aspects of the

28 interaction of T. gondii tachyzoites with host cells. At least three

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1 types of interaction between leukocytes and the parasite were
2 identified: the projection of filopodia, the formation of a tunnel-like
3 projection involving the parasite (Figure 6 E), and the invagination of
4 the leukocyte surface at the point of entry (A MacLaren et al., 2004).
5 The emission of filopodia that embrace the parasite and the glove-

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6 like tunnel around the parasite are suggestive of active invasion,
7 while the invagination of the leukocyte may correspond to

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8 phagocytosis.
9 One strategy that generates relevant new information is the

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10 use of HR-SEM to observe parasites inside host cells (Magno et al.
11 2005a). By gently scraping the surface of a cell monolayer infected

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12 with Toxoplasma gondii, parasitophorous vacuoles were exposed,
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13 revealing new aspects of the intravacuolar network assembly and
14 its mechanical role in keeping the parasite rosette stable and
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15 connected to the parasitophorous vacuole membrane (Figure 6F).

16 Previously, Schatten and Ris (2004, 2002) had observed the inner
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17 structure of T. gondii inside host cells in Epon de-embedded

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18 sections HR-SEM. Other applications of the dry scraping

19 technique were introduced by Muñiz-Hernández et al. (2011) in
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20 studies of the residual body of Toxoplasma. Using transmission

21 electron microscopy of thin sections, the presence of structures
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22 located within the parasitophorous vacuole was revealed. These

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23 structures were initially described as a tubular and fibrous material

24 (Sibley et al., 1986) and later described as a tubular network
25 (Leriche and Dubremetz, 1990; Sibley et al. 1995). HR-SEM of dry-
26 fractured cells showed that the membranous tubules seen within
27 the parasitophorous vacuole exhibited a rather uniform diameter of
28 30-35 nm (Magno et al., 2005a). This network would reinforce the
29 attachment of the parasites to the vacuolar membrane, to the

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1 residual body and to each other (Magno et al., 2005a). Using
2 HIM, the presence of several previously unknown structures
3 within the PV was revealed. The first and most prevalent
4 structure is the intravacuolar network (IVN). The quantity of
5 tubules increases gradually with the evolution of the

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6 intravacuolar development of the parasite, during which
7 successive divisions by endodyogeny take place. Figure 7 A

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8 shows three straight tubules anchored to a short tubule close to
9 the vacuolar membrane. The opposite ends lay over, but are not

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10 continuous with, the parasite surface. These three tubules have
11 about the same length and are equidistant from each other,

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12 forming a tripod. This symmetry suggests that this structure is
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13 highly stable and maintains the parasite in an appropriate
14 position. In other instances, the IVN tubules are more sinuous,
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15 suggesting the application of a lower tension (Figure 7 B). It was

16 suggested that the contrast between the straight appearances of
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17 some tubules could be the result of tension forces generated

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18 during the intravacuolar establishment of the parasite. In the

19 most abundant zone of tubules, such tension may not occur,
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20 probably because more structures are supporting the junction

21 between the parasite and the inner face of the PV. Apparently,
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22 IVN tubules do not fuse with the vacuolar membrane but

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23 establish a close attachment to it through tiny structures (Figure

24 7 C). At high magnification (250,000 times), it is clear that the
25 tubules are not smooth. Small "bumps" measuring 31.3±6 nm
26 are randomly scattered along the tubules. These bumps may
27 correspond to areas of IVN branching. The IVN tubules clearly
28 establish a bridge between the parasite surface and the
29 intravacuolar side of the PV membrane. These tubules have

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1 been described as continuous with the parasite outer membrane
2 (Magno et al., 2005b). HIM observations confirmed that, indeed,
3 many tubules were continuous with the parasite surface (Figure
4 7C), but many others only touched the parasite surface (Figure 7
5 A-C). A new feature of the intravacuolar space, as detected with

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6 HIM, was the presence of a large amount of filamentous material
7 interconnecting the IVN tubules to each other, the parasite, and the

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8 inner surface of the vacuolar membrane (Figure 7 B, C). These
9 filaments were uneven, varying in thickness from 2 to 10 nm within

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10 the same filament (Figure 7 B, C). The appearance of these
11 tubules is suggestive of an adhesive material, and a detailed look

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12 at TEM ultrathin sections reveals an amorphous content inside the
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13 PVs that is compatible with these filaments (Sibley et al., 1995).
14 Aside from these two types of filamentous structures, membranous
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15 sac-like structures, previously observed by TEM, were also seen.

2.3.2.1. The inner surface of the PV membrane

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16

17 The dry cleave technique often exposes large areas of the

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18 intravacuolar face of the PV membrane. In early vacuoles

19 (containing a single parasite), the inner surface appeared smooth,
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20 with tiny structures 100 nm long and 60 nm wide homogeneously

21 scattered. These structures may correspond to previously described
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22 invaginations of the PVM (Coppens et al., 2006). Openings in this

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23 membrane, irregular in size and distribution, were also present

24 (Figure 8 A, B). The smallest openings were pore-like, circular and
25 measured less than 10 nm in diameter, but other openings, which
26 were 100-200 nm wide, were also observed. These openings could
27 reach 300 nm in diameter. An interpretation for these observations is
28 that the newly formed PV would have many protrusions and very

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1 few and very small (10 nm or less) pores. However, the pores
2 would increase progressively in number and diameter, giving rise
3 to the observed openings and reducing the number of protrusions.
4 We speculate that these pores may be involved in the passage of
5 nutrients from the host cell into the PV. Indeed, previous studies

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6 have shown that molecules with molecular weights in the range of
7 1,300-1,900 Daltons can be transferred from the host cell

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8 cytoplasm into the PV (Schwab et al., 1994). Previous studies
9 also revealed the presence of pores with a mean diameter of 22

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10 nm distributed throughout the kidney glomerulus (Rice et al.,
11 2013). These pores were also seen by SEM using an “in-lens”

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12 detector (Gagliardini et al., 2010). These structures were
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13 considered to be “slit pores” involved in kidney blood filtration. Are
14 these pores real or artifacts induced by sample preparation,
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15 especially during critical-point drying? One can always speculate

16 that special regions of the PVM exist and could be more fragile
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17 due to their composition or association with intravacuolar or

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18 cytoplasmic structures, leading to small ruptures during the

19 processing of samples for microscopy examination. Such ruptures
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20 would be especially likely during dehydration in ethanol or even

21 during critical-point drying, in which the cells are submitted to
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22 pressures of up to 73 atmospheres. It is worthy to point out here

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23 that the pores are also seen in cryofractured samples.

24 2.3.2.2. The host cell cytoplasmic side of the surface of
25 the PV membrane
26 In a few situations, images were obtained that displayed the
27 surface of the PV membrane in contact with the host cell
28 cytoplasm (Figure 8D). Organelles, probably mitochondria, are
29 seen around the PV, as are cytoskeletal filaments (Figure 8 C, D,

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1 E and F). As magnification is enhanced, more details become
2 visible, such as endoplasmic reticulum elements networking around
3 the PV (Figure 8 E, F). As already seen via the examination of
4 ultrathin sections with TEM, the endoplasmic reticulum concentrates
5 around the PV and is very closely associated with it (Magno et al.,

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6 2005b; Sinai et al., 1997). The openings of the PV membrane
7 allowed a new view of the ER tubules, showing an intense display of

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8 fused flat cisternae 7 to 50 nm in width. It is hard to distinguish the
9 PV membranes from ER membranes, although there is clearly more

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10 than one layer of membranes below the PVM. These images
11 suggest the fusion or even the continuity of ER cisternae with the

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12 PVM (Figure 8 E, F). This observation, rather than constituting a
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13 flaw in our observations, enhances the belief that PVM growth is
14 dependent on the contribution of host cell ER membranes (de Melo
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15 et al., 1992; Melo and de Souza, 1997). Side views of the host cell
16 cytoplasm show cytoskeletal structures that have a shape and
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17 diameter consistent with microtubules, microfilaments, and

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18 intermediate filaments, which are known to participate in supporting

19 the PV (Andrade et al., 2001; Halonen et al., 1998).
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20 3. Cryo-SEM
21 The pioneer work of (Erlandsen et al., 2003) on Giardia lamblia
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22 proved that Cryo-SEM could be employed for the observation of

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23 these parasites; however, little if any new information about the

24 ultrastructure of this protozoan was obtained using this method at
25 that time. More recently, Zumthor et al. (2016) revealed the
26 presence of evenly distributed small surface indentations on the
27 actual surface of G. lamblia trophozoites that are mostly probably
28 related with elongated endocytic organelles located just below the

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1 plasma membrane. Cryo-SEM of cells infected with T. gondii was
2 used to confirm the presence of pores in the Toxoplasma gondii
3 parasitophorous vacuole membrane made by HIM (de Souza and
4 Attias, 2015). To confirm this observation, we used high-
5 resolution Cryo-SEM in which the cells were frozen, fractured,

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6 coated, and observed in a frozen state in a high-resolution SEM.
7 Supplemental Figure 3A shows that small 52 nm pores could be

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8 observed, supporting the idea that these pores are real. The two
9 kinds of openings are completely different from the openings

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10 caused by mechanical stretching. However, we cannot exclude
11 the possibility that the larger openings likely correspond to the

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12 perforin pores described by Kafsack et al. (2009) and are involved
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13 in the process of weakening the PV membrane, which is
14 necessary for subsequent parasite egress at the end of the
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15 intracellular cycle.
16 Cryo-SEM is technically challenging requiring, besides a high
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17 resolution SEM that can operate in cryo mode, that freezing

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18 devices (high pressure freezing, freeze fracture, etc) are available

19 for sample preparation. Besides, once in the SEM chamber, beam
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20 irradiation quickly degrades the exposed surfaces. These, among

21 other reasons, may have limited to date the use of Cryo-SEM.
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22 However, this approach has the potential to avoid artifacts that

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23 can be introduced by chemical fixation as well as removal of

24 sample components through the successive steps, including
25 dehydration and critical point drying. In conclusion, it may be a
26 very helpful tool for future studies of the biology of protozoan
27 parasites closer to its in vivo state, as illustrated by the images of
28 Toxoplasma gondii in Supplemental Figure 3 A and Trypanosoma
29 cruzi in Supplemental Figure 3 B.

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1

2 4. Three-dimensional reconstruction of serial images

3 by HR-SEM
4 One advance of great impact in SEM was the confirmation that it
5 was possible to obtain high-resolution images using backscattered

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6 electrons generated from the interaction of electrons accelerated at
7 relatively low voltages with the surface of materials. Adapting it to

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8 biological samples, very impressive images from the surface of
9 epoxy-embedded cells were obtained, revealing details about their

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10 inner organization that only TEM was able to show until then.
11 Supplemental Figures 4A, B and C show, respectively, the ER-Golgi

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12 complex system (De Souza, unpublished) and sub-pellicular
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13 microtubules of Trypanosoma cruzi. This frontier breaking advance
14 opened the possibility to obtain what is now considered as TEM-like
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15 images of epoxy bloc faces, an important achievement made

16 possible due to improvements in electron detectors that resulted in
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17 the generation of SEM images that resemble those obtained in TEM

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18 of thin sections. Subsequently, two new developments were

19 achieved, bringing a whole new relevance to the application of SEM
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20 to biological samples. These developments allowed the 3-D

21 reconstruction of biological structures based on serial images
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22 obtained from block surfaces by one of two processes: 1) Dual

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23 Beam FIB-SEM, that consist in nanoabrasion of the surface of the

24 sample using a source of gallium ions intercalated with the
25 acquisition of the backscattered image of the exposed surface with a
26 scanning electron beam, and 2) Microtome Serial Sections obtained
27 with a diamond knife installed inside the microscope. Each method
28 has its peculiarities: the gallium ions source used in this first
29 approach can generate abrasions as thin as 5 nm, and up to 200µm

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1 wide. However, the larger the area abraised, more time
2 consuming is the method. Usually, 20 to 30µm wide trenches give
3 the best cost/benefit/magnification/resolution return. With the
4 diamond knife 50-100nm thick slices covering larger areas can be
5 reached. However, scanning of larger areas results in smaller

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6 magnification, so, most often, the area selected for scan
7 acquisition is similar in both methods. Both strategies allow

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8 quicker and more reliable reconstruction of volumes much larger
9 than serial sections observed in TEM (Figure 9). In conclusion, the

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10 final resolution is very close to that obtained for thin sections of
11 biological materials in electron microscopy tomography (Micheva

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12 and Smith, 2007; Miranda et al., 2015; Nanguneri et al., 2012).
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13 These two approaches are described below.

14 4.1. Dual Beam Focused Ion Beam SEM (FIB-SEM)

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15 FIB-SEM shattered ancient paradigms and brought SEM to a

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16 completely new era. The main concept of this type of equipment is

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17 the combination of an electron beam and a focused ion beam (FIB)

18 in the same column. Electrons are generated from a field emission
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19 electron gun and scan the surface of the sample, generating both
20 secondary and backscattered electrons that may be captured by the
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21 corresponding detectors and converted into images. The second

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22 beam consists of gallium or xenon plasma ions that simultaneously

23 scan the surface of the sample, generating a signal that can be
24 converted into an image, and erode its surface, exposing the layers
25 below it (Heymann et al., 2006). Initially designed for micro-
26 prototyping of materials, FIB-SEM became a priceless tool for
27 biological studies. This technique produces nanometric abrasions on
28 the surface of a chemically fixed epoxy-embedded sample and

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1 sequentially captures the signal of backscattered electrons
2 generated by the exposed surface, which are derived primarily from
3 the impregnation of membranes with osmium, lead or other metals.
4 Images of series of ultrathin sections, on the order of 10-100 nm can
5 cover volumes of tens of micrometers. These series can be

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6 rendered into 3D models with a resolution similar to TEM and have
7 been widely employed in all areas of biomedical sciences. A very

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8 creative use of FIB-SEM was presented by Lemgruber et al.(2011)
9 that used the gallium ion source to mill the cystwall of cysts isolated

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10 from mice brains chronically infected with Toxoplasma gondii. As the
11 cyst matrix was exposed, a network of tubules was revealed, connecting

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12 the bradyzoites to each other and to the cyst wall.
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13 More recently, it was shown that it is possible to use the FIB
14 approach for cryopreserved samples (Vidavsky et al., 2016).
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15 4.2. Microtome Serial Sections (MSS)

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16 This second process was developed by Denk and Horstmann

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17 (2004), that called it serial block face SEM. Since the successive
18 surfaces of the block are exposed by microtome sections, it seems
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19 to us that it is more adequate to use “microtome serial section-SEM”

20 rather than “serial block face-SEM”, as used up to now. Basically, it
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21 consists in an ultramicrotome mounted inside the chamber of a field

emission scanning electron microscope. Sequentially, ~30–80 nm-
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22

23 thick sections are removed from the sample surface using a

24 diamond knife. The serial images are obtained using the same
25 principles described above (Bouwer et al., 2017; Leighton, 1981)
26 and 3-D models can be rendered.

27 These two approaches have already been recently applied for

28 several parasitic protozoa. In a pioneer work, Soares Medeiros et al.

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1 (2012) analyzed the 3D architecture of multiple erythrocytes
2 infected with Plasmodium chabaudi and determined some
3 morphometrical parameters analyzed in a 3D volume using the
4 dual beam SEM. This approach allowed the precise analysis of
5 the variety of shapes and sizes of erythrocytes, the surface area

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6 of the erythrocytes, and their membrane profiles, as well as the
7 internal structures of the parasites, including the polymorphic

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8 organization of hemoglobin-filled tubules. In addition, successive
9 images of several planes can be used to obtain stereological

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10 information that provides quantitative data. The usefulness of
11 this technique was further corroborated by studies with

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12 Toxoplasma gondii (Paredes-Santos et al, 2012), Strigomonas
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13 culicis (Loyola-Machado et al., 2017), Trypanosoma cruzi
14 (Alcantara et al., 2017; Vidal et al., 2016). In the case of T. gondii,
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15 the three-dimensional distribution of micronemes, which are

16 secretory organelles, shown to be radial in relation to the apical
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17 polar ring, while the number of rhoptries, that are secretory

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18 structures larger in size but fewer, could be precisely determined

19 in extracellular tachyzoites. FIB-SEM 3-D models revealed that,
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20 besides a variable number, not all rhoptries reach the more apical
21 portion of the parasite and that the number of rhoptries is variable
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22 (Paredes-Santos et al., 2012).

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23 Combining the FIB-SEM serial abrasion, electron tomography

24 and a cytochemical technique- parasites previously incubated with
25 horseradish peroxidase as an electron-dense tracer - Alcantara et
26 al. (2014) elucidated the ultrastructure of the cytostome-
27 cytopharynx complex of epimastigote forms of T. cruzi. In the
28 rendered 3-D models, the authors could define the function of the
29 different domains of the cytostome-cytopharynx in the

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1 internalization of macromolecules. It was shown that the cytostome
2 cytoskeleton was composed of two microtubule sets, including a
3 triplet that started underneath the membrane lining the cytostome
4 and a quartet that originated underneath the flagellar pocket
5 membrane and followed the preoral ridge before reaching the

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6 cytopharinx. The cytopharinx was revealed to be a dynamic
7 structure that undergoes remodeling processes associated with

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8 endocytic activity and the preparation of the cell for its division. The
9 same group (Vidal et al., 2016) later used a similar strategy to follow

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10 the intermediate steps of metacyclogenesis of epimastigotes of
11 Trypanosoma cruzi to trypomastigotes, emphasizing the cytostome-

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12 cytopharynx complex and adjacent structures, including the preoral
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13 ridge (Figure 9 A, B and C). The authors showed that the cytostome-
14 cytopharinx complex is disassembled during the metacyclogenesis
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15 process but retains the cytoplasmic microtubules. Zumthor et al.,

16 (2016) using FIB-SEM showed a tubular organization of the cortical
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17 vesicular system of Giardia trophozoites, thus confirming previous

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18 reconstructions made by electron microscopy tomography

19 (Abodeely et al., 2009; Lanfredi-Rangel et al., 1998).
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20

21 The second method, MSS-SEM, was used by to show that the

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22 distal tip of new flagellum of bloodstream forms of Trypanosoma

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23 brucei is embedded within an invagination of the cell body

24 membrane, designated as the groove, located alongside the
25 microtubule quartet (Hughes et al., 2013). In addition, Gluenz et
26 al.(2015) used it to study the flagellum of Trypanosoma brucei and
27 Leishmania mexicana at different stages of the cell cycle, including
28 procyclic and metacyclic forms. The authors not only showed that
29 these parasites have a highly ordered cell shape and form, with a

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1 defined positioning of internal cytoskeletal structures and
2 organelles, but they open the perspective of employing the
3 technique to dissect cell cycles in both parasites and the use of
4 three-dimensional reconstruction for future studies of mutant cell
5 lines (Figure 9 D,E). More recently, Hughes et al., (2017) used the

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6 same approach to reconstruct T.brucei at different stages of the
7 life cycle. Important data were obtained on changes that occur in

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8 the mitochondrial and Golgi complex distribution. The same
9 approach was used to show association of Plasmodium berghei

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10 endoplasmic reticulum with the plasma membrane and the
11 parasitophorous vacuole membrane during asexual replication

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12 (Kaiser et al., 2016). Another example of the application of MSS-
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13 SEM to the malaria parasite by Sakaguchi et al. (2016) who
14 analyzed the remodeling of the Maurer´s cleft and vesicles in
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15 erythrocytes infected with Plasmodium falciparum, showing that

16 these two structures are not connected to each other. Volumetric
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17 information was also obtained for the parasite during its

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18 intracellular life cycle.

19 So far, each method has its own advantages (larger area of
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20 acquisition in the case of MSS-SEM) and larger volume in Z and

21 better resolution with FIB-SEM. For sure, these two approaches
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22 will have further improvements in the years to come.

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23 5. High-resolution SEM Cytochemistry

24 The resolution level reached with field emission guns, and

25 improvement in secondary and backscattered electron detectors
26 soon was combined to classical cytochemistry and
27 immunocytochemistry. Nevertheless, this method remains quite
28 underexplored in the study of parasitic protozoa. One example are

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1 the images obtained with epimastigotes of T. cruzi previously
2 incubated in the presence of horseradish peroxidase to label the
3 endocytic pathway followed by cytochemical detection of the
4 peroxidase using diaminobenzinide-osmium, showed clearly the
5 areas that were labeled (Supplemental Figure 5). Besides, this

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6 approach can be explored to obtain 3D reconstructions of specific
7 labeled structures. Another potential avenue is classical

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8 immunocytochemical localization using gold-labeled antibodies,
9 since these particles can be visualized using both backscattered and

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10 secondary electrons and even the overlay of both images. As
11 previously mentioned and shown in Figure 1 this strategy was

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12 employed to label surface antigens in the cyst wall of Giardia
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13 duodenalis (Erlandsen et al., 1990). Another application, was
14 immunogold labeling performed after 2% NP-40 extraction of G.
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15 intestinalis trophozoites, followed by incubation in the presence of

16 monoclonal anti-α-tubulin TAT-1 and anti-actin antibodies. As
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17 described previously (Sant’Anna et al., 2005), the adhesive disk,

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18 axonemes, median body and funis microtubules were intensely

19 labeled by the TAT-1 antibody (Supplemental Figure 6). More
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20 recently, (Caldas et al., 2017) employed an anti-actin antibody to

21 show direct contact of Toxoplasma gondii with the cytoskeleton of
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22 its host cell during egress.

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23 1. Perspectives
24

25 In the last two decades, scanning electron microscopy advances

26 have reached an unprecedented level of resolution. Cleaving,
27 chemical maceration and detergent extraction of samples allow the
28 observation of intracellular structures with resolution that is now
29 quite similar to classical (80-200 kV) transmission electron

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1 microscopy. Helium ion Scanning Microscopy (HIM) has not only
2 reached amazing power resolution but high depth of field and the
3 avoidance of conductive coating of the sample constitutes an
4 advantage itself. In parallel, low vacuum and environmental SEMs
5 open the possibility to follow in real time biological phenomena, e.g.,

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6 motility and interaction of parasites and host cells. Dual beam and
7 the microtome series became a fundamental tool for the

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8 ultrastructural analysis of parasites and its host cells and are already
9 applied to parasites routinely fixed or in combination with

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10 electrondense tracers or cytochemical labels. The avoidance of
11 artifacts inherent to chemical fixation and the possibility of capturing

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12 instant events is a large path to be explored, in the years to come,
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13 with ultra-rapid freezing and Cryo-SEM. The combination of Cryo-
14 SEM and slice and view is also another tool that applied to 3D
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15 analysis either of parasites and their development and interaction

16 with host cells shall bring important discoveries in the field of
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17 protistology. Further advancements that can be foreseen/dreamed

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18 are dual beam HIM, cryo-HIM and higher resolution environmental

19 SEM.
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20

21 Appendix
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22 Development and applications of protocols for High

23 Resolution Scanning Electron Microscopy (HR-SEM)

24 In this session, we present three protocols- dry cleavage,

25 cryo-SEM and detergent extraction - that can be adapted by
26 other researchers, not necessarily working on parasitic

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1 protozoa models, for observation in high-resolution scanning
2 microscopy.
3 Protocol 1: The Dry Cleaving Method
4 In this method, the natural orientation of cells that grow in
5 monolayers is maintained. The idea of removing the plasma

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6 membrane to expose the interior of cells has been described and
7 applied to the tunicate Oikopleuradioica by Flood (1975). Although

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8 this sample was quite large compared to a cell monolayer, our group
9 transported this concept to expose the inner structure of the

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10 parasitophorous vacuole containing T. gondii, as well as the interior
11 of its host cells, by gently sticking adhesive tape to the surface of the

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12 previously fixed and critical-point dried monolayer. Immediately
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13 thereafter, the samples can be observed in a HIM or slightly coated
14 with a thin layer (5 nm) of platinum and observed by HRSEM
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15 (Magno et al., 2005a). More recently, we showed that this technique

16 is suitable for observation with greater resolution using HIM (de
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17 Souza and Attias, 2015).

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18 Comparing dry cleavage to other methods, it has some

19 advantages. The dry cleavage of suspended cells (embedded in a
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20 gelatin matrix), as described by Fukudome and Tanaka (1986), can

21 also be applied to parasites in suspension and host cells infected at
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22 various intervals; however, the fracture planes are random and

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23 usually very few cells in the composed pellet are cleaved. Another
24 approach was reported by Schatten and Ris (2004, 2002), who
25 used low-voltage FE-SEM to observe the parasitophorous vacuoles
26 of cells infected with T. gondii in epoxy-embedded samples where
27 the resin was partially etched chemically. This method has a limited
28 resolution and depth of the exposed area and does not allow the

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1 observation of membranous structures, such as the
2 intravacuolar network.
3 However, the dry cleavage technique is not suitable for
4 cells that do not adhere spontaneously to coverslips. Attempts
5 to cleave cells adhered to coverslips with poly-L-lysine were

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6 unsuccessful. The cells would adhere to the tape and be
7 removed from the coverslip without exposing their inner side.

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8 Interactions between host cells and parasites occur
9 under the desired conditions (parasite:host cell ratio, time of

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10 interaction, time of incubation, addition of drugs, etc.) over
11 sterile glass coverslips. The fixation of coverslips is followed

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12 by post-fixation in osmium, and then the coverslips (and the
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13 cells adhered to them) are dehydrated and critical-point dried.
14 Critical-point dried coverslips are mounted over aluminum
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15 stubs with double-sided conductive tape and gently scraped

16 with adhesive tape (Magno et al., 2005a) to expose the
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17 cytoplasmic face of the cell, including the parasitophorous

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18 vacuole. Then, the samples are sputtered with a 5 nm layer of

19 platinum in a Balzers apparatus and observed in a HR-SEM
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20 (we have used a JEOL 6340F FESEM at 5.0 kV and a working

21 distance of 8 mm, a FEI Helius or a Zeiss Auriga with lower
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22 voltages, smaller working distances and higher resolution).

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23 The scraped portion of the cells, which remain adhered to the

24 tape, can also be sputtered and observed if conductive tape is
25 used for scraping. Figure 10 is a flowchart of the procedure,
26 and Figure 11 shows the final steps, in which the sample is
27 placed into a montage and the monolayer is cleaved.

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1 The results obtained using this technique, both in HR-SEM
2 and HIM, are exemplified in Figures 6F (HR-SEM), 7 and 8 (HIM).
3

5 Protocol 2: Cryo-SEM

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6 The cryo-SEM method requires more specific equipment.
7 This method is based on observing cells that were quickly frozen

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8 and fractured to expose their inner structures in the frozen state.
9 Either impact on a cooled copper block (Figure 12) or high-pressure

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10 freezing apparatus can be used. Once frozen, the samples are
11 transferred to a freeze-fracture apparatus to be fractured, and the

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12 exposed surface is sublimated and sputtered with platinum to
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13 become resistant to the electron beam of a HR-SEM with a cryo-
14 attachment, with which the samples will be observed. A flowchart of
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15 the sequence is in Figure 13. We have used this method on T.

16 gondii and Trypanosoma cruzi (Supplemental Figure 3). Other
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17 researchers have applied this approach to other biological systems,

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18 such as sea urchin embryos (Walther et al., 1993), the rat pancreas
19 (Walther, 2003), and Chlamydia (Wilkat et al., 2014). Freeze-
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20 fracture of live cells for cryo--SEM observation has the potential to

21 reveal ultrastructural features, while avoiding artifacts of chemical
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22 fixation. On the other hand, there are some limitations since

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23 freezing efficiency is limited to samples of a certain size because

24 freezing by impact cannot avoid ice crystal formation more than 20
25 µm below the surface.
26 The procedure will need adaptations for each parasite. In
27 general terms, these (parasites) are grown under the usual
28 conditions, centrifuged to form a pellet that is rinsed and suspended
29 in a small volume of PBS (~200µl). Aliquots of 20 µl of the pellet

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1 over are placed over sheet of gelatin. The gelatin itself is glued
2 to filter papel over an aluminum disk. The assembly is frozen
3 by impact on a copper plate cooled to -196°C with l iquid
4 nitrogen, transferred in a Dewar cooled with liquid nitrogen to a
5 freeze-fracture device cooled to -120°C, fractured with a

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6 cooled blade, and immediately warmed to -105°C for partial
7 sublimation of water, exposing inner cell structures. The

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8 exposed surface is covered with platinum and carbon, and
9 transferred to a HR-SEM equipped with a cryo chamber.

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10 Observation is done at low (1-5 KV) voltage and 5 nm WD. A
11 flowchart of the procedure is shown in Figure 13. The

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12 equipment used in the process is shown in Figure 12 and
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13 images obtained with the technique are presented in
14 Supplemental Figures 3.
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15

16
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17 Protocol 3: Detergent extraction

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18 Detergents have been employed to expose the

19 cytoskeletons of cells for observation both by TEM and SEM.
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20 As stated earlier in this review, for each cell type, several

21 parameters, besides the choice of the detergent, must be
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22 established (Bell et al., 1988; Lindroth et al., 1992). However,

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23 all protocols followed the same rationale of detergent

24 extraction followed by chemical fixation, dehydration, critical-
25 point drying, conductive sputtering, and observation via HR-
26 SEM or HIM.
27 Detergent extraction is technically less demanding than
28 freeze fracture, although superb results were obtained by
29 Heuser and Kirschner (1980) to observe the network of

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1 cytoskeleton filaments in mammalian cells, in metal replicas of
2 quickly frozen, freeze-fractured, deep-etched cells, observed first by
3 TEM and later by FE-SEM. This technique was successfully applied
4 to the following protozoan parasites: trypanosomatids (Souto-
5 Padrón et al., 1984), Giardia lamblia trophozoites (Kattenbach et

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6 al., 1996), and Tritrichomonas foetus (Benchimol and De Souza,
7 1987). However, as said above, the execution of these protocols is

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8 far more demanding than the protocol proposed here. Combinations
9 of detergents for extracting the plasma membrane and exposing the

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10 cytoskeleton have been tried, and they worked very well in several
11 cell models, both mammalian (Svitkina et al., 1995) and protozoan

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12 (de Andrade Rosa et al., 2013; de Souza and Attias, 2015;
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13 Holberton and Ward, 1981; Sant’Anna et al., 2005). It is worth
14 noting that detergent extraction can be applied for both TEM and
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15 SEM, with few adjustments to the protocol. Detergent extraction is

16 suitable for almost any cell, except specimens carrying a cell or cyst
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17 wall (plants, fungi, and cystic forms). Nevertheless, for each type of
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18 protozoan, the concentration of the detergent, the temperature, and

19 the extraction time must be adapted. Another point worth
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20 mentioning is that, although the execution of detergent extraction

21 itself is simple, the person executing the protocol must have a
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22 “trained eye” to recognize, under the light microscope, the point at

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23 which extraction should be stopped. It can also be combined to

24 colloidal gold immunocytochemical labeling (Supplemental Figure
25 6),
26 The below conditions were successfully applied to Giardia and
27 Tritrichomonas foetus trophozoites. Grow cells (parasites) under the
28 usual conditions. Centrifuge the suspension, rinse in phosphate-

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1 buffered saline (PBS), and resuspend at a final concentration
2 of 106 cells/ml.
3 Pre-coat glass coverslips with a 0.01% poly-L-lysine (mol
4 wt 150,000–300,000, Sigma, USA) solution, allowing contact
5 with the solution for at least 15 min. Quickly and gently rinse

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6 the coverslips in distilled water and add 20 µl of the living cells
7 suspension (1x107 cells/mL). After 15 min, remove non-

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8 adhered parasites by gently rinsing in PBS, pH 7.2. For
9 cytoskeleton extraction, the cells were treated with 2% Triton

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10 X-100 and 2% NP-40 in a specially modified buffer for isolating
11 the cytoskeleton (IC buffer: 10 mM Tris Base, 2 mM EDTA, 2

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12 mM DTT, 2 mM MgSO4, 150 mM KCl, 30% glycerol, pH 7.4)
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13 for 30 min or 1 h (Palm et al., 2005). After this time, the IC
14 buffer was removed, and the whole mount was fixed in 2.5%
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15 glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, and washed

16 in PBS, pH 7.2. The sample was then post-fixed for 30 min in
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17 1% OsO4 in 0.1 M phosphate buffer, pH 7.2, dehydrated in an

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18 ascending ethanol series ending in 100% ethanol, critical-point

19 dried with CO2, and sputter-coated with a thin layer (1–2 nm)
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20 of chromium or carbon.
21 Figure 14 is a flowchart of the procedure. The results
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22 obtained are exemplified in Figures 3 and 4 (for

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23 Tritrichomonas) and 5 (for Giardia intestinalis).

24

25

26 Acknowledgements
27 This work was supported by Conselho Nacional de
28 Desenvolvimento Cientıf́ ico e Tecnológico (CNPq) [grants to MA
29 and WS];and Cientista do Nosso Estado [grants to MA and WS]

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1 from the Fundação Carlos Chagas Filho de Amparo à Pesquisa do
2 Estado do Rio de Janeiro (FAPERJ).
3

4 References

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20 Woessner, D.J., Dawson, S.C., 2012. The Giardia Median Body

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1 292–301. doi:10.1128/EC.05262-11

2 Zumthor, J.P., Cernikova, L., Rout, S., Kaech, A., Faso, C., Hehl,

3 A.B., 2016. Static Clathrin Assemblies at the Peripheral

4 Vacuole—Plasma Membrane Interface of the Parasitic

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6 doi:10.1371/journal.ppat.1005756

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7

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8 Legend to Figures
9 Figure 1: Giardia duodenalis. (A) Secondary electrons image
10
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(SEI) of the ventral surface of a trophozoite of the MR4 strain12
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11 hr after stimulation of in vitro encystment. Framed area:
12 developing individual filaments in the ventral attachment disc
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13 shown at higher magnification in SEI (B), and in backscattered

electron imaging (BEI) (C). Arrowheads: immunogold particles to
D

14

15 cystwall antigens; (f) flagellum. (from: Erlandsen et al. (1990)

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16 with permission).
17 Figure 2: HIM of extracellular tachyzoites of Toxoplasma
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18 gondii. (A) Apical end with the conoid (c) in the upright position
19 polar ring (arrowhead) and conoidal fiber pattern (thin arrow) are
C

20 seen. The surface pattern with the membrane is elevated (short

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21 arrows) point the position of the subpelicular microtubules. (B)

22 The conoid is hidden below the polar ring. The thick arrows point
23 to tubular structures of unknown nature. (C) View of the surface
24 of two intracellular parasites showing secretory (white arrow) and
25 adhesion processes between two daughter cells (inset on the
26 upper right: higher magnification of the structure shown in the

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1 rectangle). Rather than a simple vesicle, the structure has a
2 peculiar doughnut shape. In the upper left inset: two
3 tachyzoites inside a parasitophorous vacuole depicting the
4 area enlarged. (From de Souza and Attias, 2015).

5 Figure 3: HR-SEM of the pelta-axostilar system. (A) Overview of

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6 the Pelta (P) that is located in the anterior region, supporting the
7 flagellar canal and the axostyle (Ax). (B) and (C) Zoom views of

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8 the pelta-axostylar junction. Each microtubule is clearly
distinguished and they are organized in two distinct groups.

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9

10 (From de Andrade Rosa et al., 2013) .

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11 Figure 4: Cytoskeleton of T. foetus seen by super high resolution
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12 SEM. (A) and (B) two costa (purple) are seen, indicating that the
13 cell is dividing. Note the presence of an accessory filament
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14 (orange), which is originated in the anterior cell region,

15 separately from the costa. These structures have a periodic
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16 pattern and are joined together at the anterior cell region

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17 remaining associated throughout their length. (P) pelta, (Ax)

18 axostyle, (RF) recurrent flagellum, (N) nucleus, (C) filamentous
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19 network (Nt) located under the undulating membrane, which

20 connects the accessory filament (*) of the costa (C) to the
C

21 recurrent flagellum (RF). (D) striated fibers of T. foetus. The

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22 costa (purple) is seen as the thickest fiber, while the parabasal

23 filament (pink) is slightly flattened. Both structures have similar
24 periodicity. (E) cytoskeletal structures of the anterior region. The
25 microtubules of the axostyle (green), the comb (orange) that is
26 composed of small filaments connecting the costa (purple), and
27 the infra-kinetosomal body (IKB). The sigmoidal filaments (S) are
28 over the basal bodies. (From de Andrade Rosa et al., 2013)

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1 Figure 5: Detergent extracted trophozoites of Giardia
2 intestinalis observed under high resolution scanning electron
3 microscopy (A) and (C) and HIM (B) and (D). (A) General view of
4 a trophozoite, in which the ventral disk (VD) and some flagella (f)
5 are pointed. (B) The marginal plate (*) is connected with the

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6 axoneme (Ax) and the network of filaments (arrow). (C) Ventral
7 view of the ‘‘bare area’’ of the ventral disk. Two sets of

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8 microtubules (1 and 2) forming the ventral disk are observed
9 (arrows). VD – ventral disk; BC2 – banded collar 2. (D) Banded

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10 collar 2 (BC2) showing horizontal segments connected by small
11 bridges (arrowhead). These segments were continuous with the

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12 axonemes (arrow) of the left lateral-posterior (p) and caudal
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13 flagella. (A) (From Maia-Brigagão et al., 2013); (B), (C) and (D)
14 (From Gadelha et al., 2015).
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15 Figure 6: HR-SEM of interaction between parasitic protozoa

and host cells. Peritoneal macrophages and LLC-MK2 cells
D

16

17 treated with amiloride. Interaction for 2h with Trypanosoma cruzi

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18 trypomastigotes (A) and (C) and amastigotes (B), demonstrating

19 impairment of parasite entry after treatment. (D) T. cruzi
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20 amastigote enveloped by a macrophage plasma membrane

21 (arrow) after treatement with 60 mM of dynasore during 20
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22 minutes. (E) Toxoplasma gondii tachyzoite (T) invading a mouse

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23 neutrophil (N). Intermediary situation between filopodial

24 embracement and the membrane tunnel around the tachyzoite
25 (star). (F) Rosette of tachyzoites inside the parasitophorous
26 vacuole (PV) after dry scraping of the host cell. Arrows point to
27 tubules of the intravacuolar network. (A), (B) and (C) (From

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1 Barrias et al., 2012). (D) (From Barrias et al., 2010). (E) (
2 From A. MacLaren et al., 2004) .

3 Figure 7: HIM of intracellular tachyzoites of Toxoplasma

4 gondii (A) Secretion of dense granules leads to the formation
5 of the intravacuolar network (squares). Inset: higher

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6 magnification of the area in the square: Three straight tubules
7 anchored to a shorter one in the parasitophorous vacuole

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8 membrane. These three tubules form a tripod and the
extremities are apposed but not fused with the parasite

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9

10 surface. (B) The sinuous arrow runs parallel to a long and

11 wavy IVN tubule. Filamentous extensions (arrowheads) are
12
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seen linking IVN tubules. (C) Intravacuolar network seen at
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13 higher magnification. White arrowheads point to ‘‘bumps’’ that
14 may correspond to the point of origin of a new branch of the
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15 tubule. The thick black arrow points to a tubule that appears

continuous with the PVM. Small black arrowheads point to tiny
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16

17 filamentous structures that link the IVN tubule to the PVM

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18 inner surface. (From de Souza and Attias, 2015) .

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19 Figure 8: Toxoplasma gondii. HIM of intracellular parasites.

20 (A) Parasitophorous vacuole (PV) membrane. Arrowheads
C

21 point to 10 nm pores scattered over it. As the pores enlarge,

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22 they become less circular (white arrows) when compared to

23 the smaller ones (black arrows). Arrowheads point to the
24 smallest pores, and circles outline membrane protrusions
25 suggestive of invaginations. The area limited by the square is
26 enlarged in (B). Toxoplasma gondii. HIM of the relationship of
27 the PV and the host cell. (C) Three vacuoles contiguously
28 placed in a host cell. Organelles (probably mitochondria) are

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1 seen around them (m). In all three vacuoles, parasites are
2 dividing; the center one is at the end of the first endodyogeny
3 division. In the one at the lower left, daughter cells are about to
4 emerge, and on the right, the invagination at the posterior region
5 is indicative of the process. (D) The PV membrane can be seen

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6 from the cytoplasmic side. The appearance of the pores (arrow)
7 are similar to those observed in the inner surface. (E) As the

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8 pores enlarge, elements that probably belong to the endoplasmic
9 reticulum around the PV (white arrow) Appear from the inside of

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10 the vacuole. In the area marked by the rectangle, the membrane
11 layers can be distinguished, but in the area encircled, the

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12 distinction is not so clear, a suggestion of fusion of ER
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13 membranes with the PVM. The black arrow points to what
14 probably is a filament of the host cell cytoskeleton. (F) Views by
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15 HIM of the cytoskeleton around the parasitophorous vacuole.

16 Filaments of variable size associated to other filamentous
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17 structures are seen around the PV. Arrowhead points to a

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18 possible microtubule and the thin arrow points to a thin filament.

19 (From de Souza and Attias, 2015) .
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20
C

21 Figure 9: (A) 3D model and corresponding Z-slices (B–C)

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22 from FIB-SEM of the cytostome-cytopharynx area of

23 metacyclogenesis intermediate Ib cell of Trypanosoma cruzi.
24 Nucleus (N), blue; Kinetoplast (K), green; Flagellum (F), yellow;
25 Flagellar pocket (FP), white; Golgi complex (Gc), brown; Preoral
26 ridge (POR), purple; Cytopharynx (Cy), pink; Reservosomes (R),
27 red. Scale bars, 200 nm. (D)(E) 3D model from Microtome Serial
28 Section of bloodstream form Trypanosoma brucei. (D) A single

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1 slice (100 nm thick) from an MSS-SEM dataset, illustrating
2 organelles and cytoskeletal structures of the cell. (E) Surface
3 volume rendering of the MSS-SEM dataset containing a whole
4 cell with a transverse slice from the dataset to illustrate the
5 rendering process. Mitochondrion (M)—green; nucleus (N)—

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6 blue; old flagellum (F)—purple; new flagellum (arrow in B)—
7 red; glycosomes (G)—orange; acidocalcisomes (A)—white;

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8 vesicles—yellow. Scale bar: 500 nm. (A-C: (From Vidal et al.,
9 2016b); D-E (From Gluenz et al., 2015b).

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10 Legend to Appendix Figures

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11 Figure 10: Flowchart and timing for protocol 1 (dry cleavage).
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12 Figure 11: Montage and scraping of a monolayer of cells. (a)
13 Coverslip with cell monolayer after critical-point drying. The cells are
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14 on the dull side of the coverslip. (b) Place a disc of double-coated

15 adhesive conductive tape on the appropriate SEM stub. (c) Adhere
D

16 coverslip with sample to the stub. (d) Touch the surface of the
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17 coverslip with a second stub with adhesive tape. (e) The scraped
18 portion of the sample remains on the second stub (white
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19 arrowhead). A darker area on the coverslip corresponds to the

20 scraped area (black arrowhead). (f) Higher magnification of the
C

21 coverslip where the scraped area looks darker (black arrow) than
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22 the unscraped surface (white arrow).

23 Figure 12: Slam freezing sequence. (a) view of the slam freezing
24 machine in the resting position. The arrow points to the detachable
25 head. (b) sample holder composed of an aluminum disk with a filter
26 paper disk glued to it and a disk of gelatin over it. (c) sample holder
27 attached to the detachable head. (d) application of the sample to the
28 sample holder. (e) (f) placement of the detachable head in the slam

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1 freezing device. (g) the sample is frozen by impact over a copper
2 plate cooled to -196°C with liquid nitrogen.
3 Figure 13: Flowchart for protocol 2 (fracture and cryo-SEM).
4 Figure 14: Flowchart of procedures for protocol 3 (detergent
5 extraction).

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6 Legends to supplemental Figures:
7 Supplemental Figure 01: Surface views of T.cruzi Y strain

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8 epimastigote forms sites of entry by high resolution scanning
9 electron microscopy. The area between the cytostome and the

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10 flagellar pocket is a special membrane domain displaying a
11 rugous texture. From (Vatarunakamura et al., 2005).

12
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Supplemental Figure 2: Cytoskeleton of the pseudocyst of T.
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13 foetus as seen by high resolution SEM. (A) The axostyle (Ax-
14 green) surrounds the nucleus (N-red), the flagella (F) and the U
M

15 shaped costa (C-purple). (B) fragmented axostyles (Ax-green)

and the curved costa (C) around one of the axostyles. (C) Higher
D

16

17 magnification of the U-shaped costa. From (de Andrade Rosa et

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18 al., 2015).
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19 Supplemental Figure 3: Cryo-SEM (A) Toxoplasma gondii

20 inside the PV.The inner surface of the PVM has scattered, pore-
C

21 like openings of diverse diameters. Arrowheads indicate

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22 rhoptries in a cleaved parasite. (B) Trypanosoma cruzi

23 epimastigotes. Two cells were longitudinally cleaved exposing
24 the nucleus (N), Golgi complex (CG) and nuclear pores
25 (arrowheads). (A) (From de Souza and Attias, 2015), (B)
26 Courtesy of Kildare Rocha de Miranda.

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1 Supplemental Figure 4: A) and (B): FIB slices of Trypanosoma cruzi.
2 (A) The Golgi complex (GC), glicosomes (g) and mitochondrion (m)
3 can be seen. (B) View of the nucleus (N), cross view of the flagellum
4 (F). Two sets of microtubules are pointed in the inset: the preoral
5 ridge (orange) and cytostome (blue). (C) Partial reconstruction of

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6 epimastigote forms of Trypanosoma cruzi from serial slices obtained
7 with FIB-SEM. (B) (From Vidal et al., 2016b).

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8 Supplemental Figure 5: FIB-SEM micrograph of
9 Trypanosoma cruzi epimastigotes that had uptaken

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10 horseradish peroxidase (HRP) for 5 min at 28°C. The HRP-
11 DAB immunocitochemistry allowed clear distinction between

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12 the endocytic compartments of the parasite and the other
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13 unstained compartments that do not participate of the
14 endocytic pathway. Bars 1µm. Courtesy Carolina Alcântara
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15 and Narcisa Cunha.

Supplemental Figure 6: Giardia trophozoites immunolabeled

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16

17 for α-tubulin observed using the backscattered electrons detector

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18 (BEI). Labeling is seen in the flagella (arrowhead) and ventral

19 disk (VD). (From Gadelha et al., 2015).
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Graphical Abstract

Compared to the first SEM, field emission electron and Helion ion scanning electron
microscopy exponentially enhanced the resolution of these instruments. Recent progress in
the study of the ultrastructure of protozoan parasites is reported in this review, as well as
some protocols for sample preparation. In this micrograph, the cystoskeleton of Giardia
intestinalis was exposed by detergent treatment and observed by High Resolution Scanning
Electron Microscopy. Bar: 200 nm.(from: Rocha Gadelha et al., 2015)

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Highlights

Protist ultrastructure research much benefited by SEM improvements in resolution and

detectors.

Chemical and cryofixation methods and protocols are presented and discussed.

Three dimensional reconstruction with dual beam SEM opens a new era of possibilities.

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