There are many reasons to examine human cells and tissues

under the microscope. Medical and biological research is

underpinned by knowledge of the normal structure and
function of cells and tissues and the organs and structures that
they make up. In the normal healthy state, the cells and other
tissue elements are arranged in regular, recognizable patterns.
Changes induced by a wide range of chemical and physical
influences are reflected by alterations in the structure at a
microscopic level, and many diseases are characterized by
typical structural and chemical abnormalities that differ from
the normal state. Identifying these changes and linking them to
particular diseases is the basis of histopathology and
cytopathology, important specializations of modern medicine.
Microscopy plays an important part in haematology (the study
of blood), microbiology (the study of microorganisms
including parasites and viruses), and more broadly in the areas
of biology, zoology, and botany. In all these disciplines,
specimens are examined under a microscope.
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Microscopy
There are many different forms of microscopy, but the one most commonly
employed is “brightfield” microscopy where the specimen is illuminated
with a beam of light that passes through it (as opposed to a beam of
electrons as in electron microscopy). The general requirements for a
specimen to be successfully examined using brightfield microscopy are:
 That the cells and other elements in the specimen are preserved in a
“life-like” state (this process is called “fixation”)
 That the specimen is transparent rather than opaque, so that light can
pass through it
 That the specimen is thin and flat so that only a single layer of cells is
present
 That some components have been differentially coloured (stained) so
that they can be clearly distinguished

Preparation options
Because of the microscopy requirements, options for preparing specimens
are limited to: 

 Whole-mounts, where an entire organism or structure is small enough or

thin enough to be placed directly onto a microscope slide (e.g. a small
unicellular or multicellular organism or a membrane that can be
stretched thinly on to a slide)
 “Squash” preparations, where cells are intentionally squashed or
crushed onto a slide to reveal their contents (e.g. botanical specimens
where cells are disrupted to reveal chromosomes)
 Smears, where the specimen consists of cells suspended in a fluid (e.g.
blood, semen, cerebro-spinal fluid, or a culture of microorganisms), or
where individual cells have been scraped brushed or aspirated (sucked)
from a surface or from within an organ (exfoliative cytology). Smears
are the basis of the well-known “Pap test” that is used to screen for
cervical cancer in women.
 Sections, where specimens are supported in some way so that very thin
slices can be cut from them, mounted on slides, and stained. Sections
are prepared using an instrument called a “microtome”.
Of these options only whole-mounts and sections preserve the structural
relationships between individual cells and extracellular components.
Smears and squash preparations provide detail about individual cells and
relative cell numbers, but structural relationships are lost. The preparation
of sections is the most technically complicated of these methods as it
requires specialized equipment and considerable expertise. The
microscopic examination of sections by a pathologist forms the corner
stone of cancer diagnosis. Although the methodology for preparing
sections from both animal and plant material is similar, the following
description relates to animal (human) tissues.

Section preparation
Most fresh tissue is very delicate, easily distorted, and damaged. Thus, it is
impossible to prepare thin sections (slices) from it unless it is supported in
some way whilst it is being cut. Usually, the specimen also needs to be
preserved or “fixed” before sections are prepared. Broadly, there are two
strategies that can be employed to provide this support.
1. The tissue can be rapidly frozen and kept frozen while sections are cut
using a cryostat microtome (a microtome in a freezing chamber). These are
called “frozen sections”. Frozen sections can be prepared very quickly and
are therefore used when an intra-operative diagnosis is required to guide a
surgical procedure or where any type of interference with the chemical
makeup of the cells is to be avoided (as in some histochemical
investigations).
2. Alternatively, specimens can be infiltrated with a liquid agent that can
subsequently be converted into a solid that has appropriate physical
properties that will allow thin sections to be cut from it. Various agents can
be used for infiltrating and supporting specimens, including epoxy and
methacrylate resins, but paraffin wax-based histological waxes are the
most popular for routine light microscopy. This produces so-called
“paraffin sections”. These sections are usually prepared with a “rotary”
microtome. “Rotary” describes the cutting action of the instrument. In all
histopathology laboratories, paraffin sections are routinely prepared from
almost every specimen and used in diagnosis.
The following paragraphs describe the major steps in preparing paraffin
sections. These steps generally dictate the layout and workflow in large,
specialist histopathology laboratories where hundreds of specimens are
handled every day.
Figure 1: A diagnostic section being prepared with a cryostat microtome. The section, which has been cut from snap-frozen
tissue, is being picked up onto a warm slide where it will be immediately fixed and stained.
Figure 2: A rotary microtome being used to cut paraffin sections. In the foreground, a ribbon of sections is being “floated
out” ready for mounting on a microscope slide.

Specimen reception
Specimens received for histological examination may come from a number
of different sources. They range from very large specimens or whole
organs to tiny fragments of tissue. For example, the following are some of
the specimen types commonly received in a histopathology lab.

 Excision specimens (surgical biopsies), where whole organs or affected

areas are removed at operation
 Incisional biopsy specimens, where tissue is removed for diagnosis
from within an affected area
 Punch biopsies, where punches are used to remove a small piece of
suspicious tissue for examination (often from the skin)
 Shave biopsies, where small fragments of tissue are “shaved” from a
surface (usually skin)
 Curettings, where tissue is removed in small pieces from the lining of
the uterus or cervix
 Core biopsies, where a small tissue sample is removed using a special
needle, sometimes through the skin (percutaneously)
Specimens are usually received in fixative (preservative) but sometimes
arrive fresh and must be immediately fixed. Before specimens are accepted
by a laboratory, the identification (labeling) and accompanying
documentation will be carefully checked, all details recorded, and
“specimen tracking” commenced. It is vital that patient or research
specimens are properly identified, and the risk of inaccuracies minimized.
Figure 3: A fresh, unfixed specimen after surgical removal. To prevent degeneration or drying out, the specimen should be
fixed as soon as possible.
Fixation
Fixation is a crucial step in preparing specimens for microscopic
examination. Its objective is to prevent decay and preserve cells and tissues
in a “life-like” state. It does this by stopping enzyme activity, killing
microorganisms, and hardening the specimen while maintaining sufficient
molecular structure to enable appropriate staining methods to be applied
(including those involving antigen-antibody reactions and those depending
on preserving DNA and RNA). The sooner fixation is initiated following
the separation of a specimen from its blood supply, the better the result will
be. The most popular fixing agent is formaldehyde, usually in the form of a
phosphate-buffered solution (often referred to as “formalin”). Ideally,
specimens should be fixed by immersion in formalin for six to twelve
hours before they are processed.
Figure 4: A surgical specimen fixing in formalin and ready for grossing. Note that there is a generous volume of fixative
compared to the size of the specimens. A cassette that will contain the specimen during processing has already been printed
with patient identifiers.
Grossing
Grossing, often referred to as “cut-up”, involves a careful examination and
description of the specimen that will include the appearance, the number of
pieces, and their dimensions. Larger specimens may require further
dissection to produce representative pieces from appropriate areas. For
example, multiple samples may be taken from the excision margins of a
tumour to ensure that the tumour has been completely removed. In the case
of small specimens, the entire specimen may be processed. The tissues
selected for processing will be placed in cassettes (small perforated
baskets), and batches will be loaded onto a tissue processor for processing
through to wax.
Figure 5: This surgical specimen of stomach has been fixed in formalin. Slices about 4mm thick will now be taken from
appropriate areas and placed in the labeled cassettes for processing.

Processing
Where large batches of specimens are processed for paraffin section
preparation, automated instruments called “tissue processors” are used.
These instruments allow the specimens to be infiltrated with a sequence of
different solvents finishing in molten paraffin wax. The specimens are in
an aqueous environment to start with (water-based) and must be passed
through multiple changes of dehydrating and clearing solvents (typically
ethanol and xylene) before they can be placed in molten wax (which is
hydrophobic and immiscible with water). The duration and step details of
the “processing schedule” chosen for a particular batch of specimens will
depend on the nature and size of the specimens. Schedules can be as short
as one hour for small specimens or as long as twelve hours or more for
large specimens. In many labs, the bulk of processing is carried out
overnight. At present, there is considerable pressure on laboratories to use
processors capable of rapid processing in an effort to improve workflow
and reduce turnaround times.
Figure 6: A tissue processor being loaded with a basket of cassettes containing tissue specimens for processing. Details of the
processing steps and the schedule are shown on the screen of the processor.

Embedding
After processing, the specimens are placed in an embedding centre where
they are removed from their cassettes and placed in wax-filled molds. At
this stage, specimens are carefully orientated because this will determine
the plane through which the section will be cut and ultimately may decide
whether an abnormal area will be visible under the microscope. The
cassette in which the tissue has been processed carries the specimen
identification details, and it is now placed on top of the mold and is
attached by adding further wax. The specimen “block” is now allowed to
solidify on a cold surface, and when set, the mold is removed. The cassette,
now filled with wax and forming part of the block, provides a stable base
for clamping in the microtome. The block containing the specimen is now
ready for section cutting.
Figure 7: Processed tissue blocks are embedded into wax molds and placed on a cold plate to cool and solidify.

Sectioning
Sections are cut on a precision instrument called a “microtome” using
extremely fine steel blades. Paraffin sections are usually cut at a thickness
of 3 - 5µm, ensuring that only a single layer of cells makes up the section
(a red blood cell has a diameter of about 7µm). One of the advantages of
paraffin wax as an embedding agent is that as sections are cut, they will
stick together edge-to-edge, forming a “ribbon” of sections. This makes
handling easier.
Sections are now “floated out” on the surface of warm water in a flotation
bath to flatten them and then picked up onto microscope slides. After
thorough drying, they are ready for staining.
Figure 8: A ribbon of sections being cut from a paraffin block using a rotary microtome. Note that the sections, which are
4µm thick (4/1000 of a millimetre), show little distortion or disruption.
Figure 9: A paraffin section being mounted on a microscope slide after being floated out on warm water to flatten it.

Staining
Apart from a few natural pigments such as melanin, the cells and other
elements making up most specimens are colorless. In order to reveal
structural detail using brightfield microscopy, some form of staining is
required. The routine stain used universally as a starting point in providing
essential structural information is the hematoxylin and eosin (H&E) stain.
With this method, cell nuclei are stained blue, and cytoplasm and many
extra-cellular components in shades of pink. In histopathology, many
conditions can be diagnosed by examining an H&E alone. However,
sometimes additional information is required to provide a full differential
diagnosis, and this requires furthermore specialized staining techniques.
These may be “special stains” using dyes or metallic impregnations to
define particular structures or microorganisms, or immuno-histochemical
methods (IHC) involving the location of diagnostically useful proteins
using labeled antibodies. Molecular methods such as in-situ hybridisation
(ISH) may also be required to detect specific DNA or RNA sequences.
These methods can all be applied to paraffin sections, and in most cases,
the slides produced are completely stable and can be kept for many years.
After staining, the sections are covered with a glass coverslip and are then
sent to a pathologist who will view them under a microscope to make an
appropriate diagnosis and prepare a report.
Figure 10: The H&E stain. This is a microscopic image (micrograph) of a paraffin section of the wall of a human appendix
taken using brightfield microscopy. Cell nuclei are stained blue, while smooth muscle, collagen, and other components are
stained in shades of pink. The large clear spaces belong to fat cells, the fat having been dissolved out during processing.

Figure 11: A rack of paraffin sections being loaded onto an automated stainer for H&E staining. This instrument will stain the
sections. The adjacent automated glass coverslipper will apply glass coverslips to the surface of the sections to preserve them
and provide optimal optical conditions for microscopy.
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About the author

Geoffrey Rolls, BAppSc, FAIMS
Geoffrey Rolls is a Histology Consultant with decades of experience in the
field. He is a former Senior Lecturer in histopathology in the Department
of Laboratory Medicine, RMIT University in Melbourne, Australia.