Assignment #

Company:
Thermo Fisher Scientific

Team Number:
11B

Name(s) Coach(es):
Emlyn Stephens

Names of all team Jor Frencken

members: Jels van Arragon
Yu Xia de Jong
Yuran van der Graaf
Samuel da Silva Corrêa
Mark van der Gun

Number of words 1:
2904

1
This will be checked and includes all words after the front page, all words in tables and all words in figures.
Excluded are words on front page and words in references. All text after the word limit is reached will not be read
and graded. No tricks and discussion please.
 C ONTENTS

I Introduction 2
I.1. Phase shift . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
I.2. Scattering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
I.3. Interference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

II Working principles of TEM 2

II.2. Phase plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

III Volta phase plate 4

III.1. Working of the Volta phase plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
III.2. The Volta phase plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
III.3. Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
III.3. Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

IV Laser phase plate 4

IV.1. What is a laser phase plate? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
IV.2. Working of the Laser phase plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
IV.3. Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
IV.4. Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

V Pulsed laser phase plate 6

V.1. The Pulsed laser phase plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
V.2. Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
V.3. Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

References 7

I. INTRODUCTION
An electron microscope (EM) uses electrons to make
images of very small objects. There are multiple kinds of
electron microscopes, but in this report, only the trans-
mission electron microscope (TEM) will be explained. To
fully understand electron microscopy, one needs to un-
derstand that electrons can be both particles and waves,
depending on the circumstances.
An ordinary light microscope uses photons (light
waves). When imaging very small objects, the wave-
length of visible light will be too big to measure the ob-
ject. The image will become blurred. In contrast, an elec-
tron microscope uses electrons that have a way smaller
wavelength. That makes it possible to image smaller ob-
jects [1].
Figure 1. A representation of the Rayleigh Criterion, showing
the way two peaks combine to form one. The point at which
the peaks are closest, but still resolvable is called the Rayleigh
Criterion [2].
This “blurring” when using a light microscope can be
explained by figure 1. An image is formed by putting tiny
elements together called pixels. Every tiny light wave
represents a pixel of the image. When two waves come
too close to each other, they will form one bigger wave.
Therefore, blurring two pixels into one. The only way to
resolve this is by making the wave more narrow, in other
words, using a shorter wavelength [3].
So an electron microscope uses electrons to image sam-
ples, which have a shorter wavelength and carry more
energy than light used in regular microscope. To pre-
vent damage from the electron beam, biological samples
imaged with TEM need to be suspended in a transpar-
ent material. Cryo-electron microscopy (Cryo-EM) uses
samples that have been rapidly frozen in water [4]. The
freezing happens so fast that the water does not have time
to form ice crystals, which would interfere with the elec-
tron beam. Despite the protection from the ice, the ex-
posure of the sample to the electron beam should still be
limited, or else the sample will be damaged. This causes
Cryo-EM to have an inherently low signal to noise ratio.
Phase plates are a component of a transmission electron
microscope that offer a solution to this problem, by in-
creasing the signal to noise ratio [5]. To understand how
2
phase plates work, it is necessary to establish some key
concepts relating to the way an EM forms an image.
I.1. Phase-shift
An electron wave can undergo two kinds of phase-
shifts. One comes from a difference in path length, while
the other comes from a force or a change in energy [1].
To explain the phase-shift from a difference in path
length we are going to use the analogy of waves moving
through the ocean. Two identical waves are going to the
same point, but wave A has to travel a greater distance
than wave B. When wave A reaches the point at its peak,
wave B might reach that same point at its valley. This
difference between these waves is the phase-shift. Due to
this phase-shift, phase contrast can be created.
I.2. Scattering
In the process of electron microscopy, electron waves
get fired at the sample one at a time. Some parts of
the wave will pass right through without interacting with
it. We call these unscattered electrons. The other parts
collide with the sample and become scattered, they come
out at different angles than the ones that passed through.
This angle will result in a difference in path length [3].
I.3. Interference
When two different waves come in each other’s proxim-
ity, they will interfere. The interference of two waves can
be seen as these two waves combining into one. When
both peaks line up, one bigger peak will be formed. When
both valleys line up, one bigger valley will be formed.
This is called constructive interference. When a peak
and valley line up, they will cancel each other out. This
is what we call destructive interference. This interference
can be nicely demonstrated by throwing two small rocks
in a still pond. Both rocks will create ripples in the pond.
These ripples will interfere. This principle can be seen in
figure 2.
II. WORKING PRINCIPLES OF TEM
The microscope emits an electron that flies towards
the sample. When this electron hits the sample, some
parts of the electron wave can pass right through, while
others interact with the sample. Their velocity decreases
due to this interaction, resulting in a phase-shift. This
phase-shift is 90 degrees compared to the unscattered
electrons, meaning that the scattered waves lag behind.
This phase-shift results from a loss in energy when the

Figure 2. This image shows the way waves from two sources
interfere, depending on the difference in path length [6].
electrons collide. An additional phase-shift is created by
the longer trajectory of the scattered electrons. [3].
To be able to form a clear image, there needs to
be enough contrast. Contrast is the difference between
light and dark, or when using colours, between multiple
colours. An electron microscope has two kinds of con-
trast: amplitude contrast and phase contrast. Amplitude
contrast (see figure 3) is dependent on the thickness and
density of the sample. The denser or thicker the sam-
ple at a specific point, the fewer electrons can make it
through and still have enough energy to create a part of
the image. That causes some darker and lighter spots on
the image.
Amplitude Contrast
Electron emitter
Sample
Lens
Phase plate
Image plane
Figure 3. A visual representation of amplitude contrast due to
the interaction of electrons with the sample. The blue beams
are the ones reacting with the sample. These get diffracted or
lose energy due to interaction with the sample, thus having a
smaller total amplitude. The red beams do not interact with
the sample. By keeping all of their energy, these will have
a bigger total amplitude [3]. (This image is created by the
authors using Adobe Illustrator.)
Phase contrast is a bit more advanced but can be ex-
plained by looking at the electron as a wave. When the
3
incoming wave hits the sample, many different parts of
the wave will scatter in different directions. When all
these parts go through the lens, they are focused on a
single point. Here, the whole wave recombines again [3].
Because of the scattering angle, some parts travel a longer
distance than others. That causes a difference in phase
between all the parts of the wave, so when they recom-
bine they will interfere. The magnitude of the phase-shift
determines if the waves interfere constructively or de-
structively [7]. Constructive interference makes the wave-
amplitude bigger, while destructive interference makes
the amplitude smaller. When the wave-amplitude de-
creases, the intensity decreases, leading to a darker con-
trast of the microscope image. Constructive interference,
on the other hand, leads to a higher intensity and thus,
to lighter spots on the image. The working principles of
phase contrast can be seen in figure 4.
Phase Contrast
Electron emitter
Sample
Lens
Phase plate
Image plane
Figure 4. The diffraction of electron beams by hitting the
sample. The greater the angle of diffraction, the longer the
path length will be. When the waves recombine, they will be
out of phase and interfere, creating phase contrast [3]. (This
image is created by the authors using Adobe Illustrator.)
Both phase- and amplitude contrast will be used to cre-
ate a clear image with the electron microscope. Ampli-
tude contrast, like the name suggests, contributes most
to the amplitude of the image. Phase contrast has a
smaller amplitude. Thereby it contributes more to the
small details. To create a detailed image, phase contrast
needs to be optimal. For this purpose phase plates are
used [8].
II.1. Phase plates
Phase plates are electron-optical instruments in mi-
croscopes used to increase contrast when imaging weak
phase objects by affecting the phase contrast. To do this,
the phase plate shifts the phase of the scattered electrons

to 90 degrees. The closer the phase-shift is to this angle,
the clearer the image at that specific point [7]. A perfect
phase plate would create an image with perfect visibility
at every point.
III. VOLTA PHASE PLATE
III.1. Signal to noise ratio
As mentioned previously, traditional Cryo-Em suffers
from having a low signal to noise ratio, which might cause
a loss of detail in the final image [9]. Signal to noise ratio
can be thought of in the following way: imagine talking
over the phone while you are standing near a highway.
The person on the other side of the line will hear the
loud sound of the cars on your end, which is the noise.
You want to reduce the noise as much as possible, or the
other person might not be able to hear you. In electron
microscopy (EM), noise also is unwanted. Recently a
solution to this problem has been discovered, the Volta
Phase Plate (VPP). Results in a study by Fukuda et al.
[9] show that “the VPP improves contrast significantly
and consequently the signal-to-noise ratio”.
III.2. The Volta phase plate
The VPP is a thin film made from amorphous car-
bon, which means that the atoms in the material have
a very irregular structure. The film is placed between
the sample and the sensor of the EM. The film is heated
to about 200 °C. The electron beam causes a so-called
“Volta-potential” on the plate, which in turn adds addi-
tional phase-shift to the scattered electron [5]. The VPP
replaces other types of phase plates, which also uses a
thin carbon film, but with a small hole in the middle.
A big drawback of this type of plate is that the electron
beam and the hole must be lined up perfectly.
III.3. Advantages
The Volta Phase Plate has, compared to other phase
plates, a long service life. A study from Danev et al.
[10] states: “The VPP has a long service life and has
been used for more than six months without noticeable
degradation in performance”. In the VPP, exact centring
of the electron beam is not necessary, the phase-shift can
be created at any place on the plate. Consequently, this
allows for automatic data collection. No other Phase
plate is able to do those two things; create a phase-shift at
any point on the plate and have automatic data collection
[11].
When the VPP is used with a small amount of defo-
cus, it makes the acquisition and analysis of the data a
lot easier [12]. Defocus is an imaging technique where
4
Figure 5. The figure shows a simulation of a portrait of Frits
Zirnike, who invented this technique of TEM, as if it were
imaged by an electron microscope. The first image uses no
defocus or phase plate, the second image uses some defocus,
and the final image uses the Volta Phase Plate without defo-
cus [5]. It can be seen that the VPP significantly improves
the image contrast. (This image is adapted from a paper by
Danev and Baumeister, 2017.)
the sensor is not exactly in the focal point of the lens. In
normal photography, this would be bad, since it causes
the image to look blurry. But in TEM it actually in-
creases contrast and the signal to noise ratio. The effect
of this can be seen in figure 5.
Because of this, VPPs have superior resolution over
traditional methods, a 2018 study by von Loeffelholz
et al. [13] shows that the VPP was able to reach a
3.2Å (0.32 nanometers) resolution with 13,469 particles
whereas traditional methods could reach a 3.1Å resolu-
tion with 35,717 particles. You can imagine how small
1Å is by thinking that one Å is the width of a pencil tip,
and a meter the size of the earth [14].
III.4. Disadvantages
It is, however, not fully understood how these phase
plates work [10]. This makes it unclear whether there is
much room for improvement in the technique.
The VPP, despite being very thin, scatters some elec-
trons causing a loss in signal, and thus image quality [12].
This causes the VPP to not be entirely predictable, and
to sometimes be unreliable during usage [15]. This prob-
lem is caused by the fact that a solid piece of material is
put in the electron beam’s path. The slightest imperfec-
tions in the material can cause a lot of noise in the final
image.
IV. LASER PHASE PLATE
IV.1. The Laser phase plate
A laser phase plate is, unlike the name suggests, not a
plate. The laser phase plate consists of a high-intensity

laser inside the microscope that is trapped in an opti-
cal cavity. An optical cavity is a cavity/pocket with a
certain arrangement of mirrors where light bounces back
and forth. In the focal point of the cavity, which is the
point where all the light rays come together after they
pass through the lens, electrons can feel the oscillating
field of the light [16]. (The oscillation can be seen as a
spring extending and contracting, where the same move-
ment will repeat many times each second.) When they go
through this focal point, the electrons undergo a phase-
shift, which improves the contrast of the image.
IV.2. Working of the Laser phase plate
The idea behind the laser phase plate is based on the
interaction between electrons and light. The photons
from the laser create an electromagnetic field. At the
focal point of the laser, all the intensity comes together
in a very tiny area, causing a peak in this field. When
the electrons go through this focal point, they will feel
a repulsive force. This force will cause the phase-shift.
This setup can be seen in figure 6. The photon density
in the light beam is proportional to the phase-shift of
an electron. Additionally, the electron phase-shift is also
equivalent to the time interval that the electron remains
in that field [16].
Figure 6. Schematic representation of the setup (with LPP):
A high-intensity (standing) laser wave is used as a phase plate.
It is positioned between two mirrors and brought into the
path of the electron beam. In the back focal plane, the elec-
tron diffraction pattern is created, which is then enlarged and
transferred to the section of the TEM column holding the
optical cavity. There, the unscattered electron beam goes
through the laser wave. Finally, the scattered (phase-shifted)
and unscattered electron beams come together in the image
plane [17].
The laser phase plate uses a so-called resonant optical
cavity to decrease the laser power requirements. The op-
tical cavity has a specific length that allows the laser to
5
reflect the exact same way it came, creating a ‘standing
wave’, a wave that oscillates in a constant position. By
reflecting many times, the power of the laser will be re-
cycled. This process amplifies the intensity of the light
at the focal point of the cavity [18].
IV.3. Advantages
Using the laser phase plate, macro-molecules that are
currently too small for cryo-EM analysis can be depicted
by improving the resolution [19].
Contrary to other micro-fabricated phase plates, the
laser phase plate does not have any materials in the (elec-
tron) beam path. Therefore, blurred and distorted im-
ages can be avoided, as the required optical elements for
the laser can be sufficiently far away. Besides, the laser
phase plate has a negligible electron loss, making elec-
tron microscopy more productive as it does not generate
a partial loss of signal [8].
Moreover, the laser phase plate is very reliable, as it
can hold off an indefinite exposure to the electron beam,
making them last way longer than other phase plates.
Because there are no interruptions due to replacing a
phase plate that had aged, the laser phase plate will make
electron microscopy more efficient [20].
Another advantage of the laser phase plate is that it
offers precise control of the phase-shift. By controlling
the phase-shift, the contrast of the final image can be
controlled as well. This happens using the phase contrast
function, a function that represents how much contrast
is transferred into the image. [4] The idea of the phase
contrast function can be seen in figure 7.
contrast
1
spatial
frequency
-1
Figure 7. Representation of the phase contrast transfer func-
tion. When the electrons go through the lens of the micro-
scope, they will all be scattered at a different angle, result-
ing in different phase-shifts. This makes the electrons travel
different path lengths, which then result in a different con-
tribution to the contrast. The different, coloured dots in the
image represent the different (scattered) electrons and their
contribution to the image contrast, where 1 is full contrast
and -1 is low contrast.
The control of the phase-shift and its contrast transfer
can be seen as an orchestra. In an orchestra, different in-
struments are playing different notes. For instance, the

violins are playing high notes. Then there are the cel-
los, playing the medium notes and then there are the
low notes, played by the basses. When you are in the
audience listening to the orchestra, you can hear all of
the notes combined. That would be like a perfect mi-
croscope, where all of the particles are transferred in full
contrast.
Unfortunately, this is not the case in current micro-
scopes; not all components of the signal are transferred
into the final image. Some of them are fully transferred,
while others are silenced and others are transferred with
opposite contrast. In the orchestra analogy, this would
be like putting a microphone above the cellos so that you
can hear them fully, but not microphoning the violins
or the basses so that you do not hear them at all. The
contrast transfer function tells us how much each of the
components arrives at the image. Therefore, controlling
the contrast transfer function is like placing the micro-
phones at different positions in an orchestra, controlling
which instruments you hear strongly and which are si-
lenced.
IV.4. Disadvantages
One of the main disadvantages of the laser phase plate
is that it is questionable if it will fit into current TEMs.
Therefore, TEM modifications are needed. These ad-
justments include a stable, high power laser field inside
the TEM (just as shown in figure 6 which automatically
aligns itself in the centre of the electron beam. When the
cavities of the TEM are used at high power, the align-
ment of the cavity and its elements (like the mirrors)
slowly drift over time. Therefore, the elements inside the
TEM must be properly stabilised.
Another technical issue is that the laser requires a lot
of power to reach the desired phase-shift of 90 degrees
[8].
V. PULSED LASER PHASE PLATE
V.1. The Pulsed laser phase plate
The laser phase plate (LPP) and the pulsed laser phase
plate (PLPP) are quite similar. The main difference is in
the name. The PLPP uses a pulsed laser beam instead
of a continuous beam. The difference is that the pulsed
beam can charge up in between pulses [21].
V.2. Advantages
Because the Pulsed laser phase plate can charge up in
between pulses, it can produce much higher peak intensi-
6
ties while maintaining the same average power consump-
tion [22]. This can be seen in figure 8.
Figure 8. The difference between the average power level of a
pulsed laser in comparison to the peak energies. High energy
levels and thus, intensities can be achieved while still main-
taining a relatively low average energy consumption(power)
by using a pulsed output. [22]
That is also the reason that it does not need the opti-
cal cavity to amplify the beam. The optical cavity was
needed in the LPP to reach the higher intensity required
to get the desired phase-shift. When you use a pulsed
beam, however, these intensities can be reached without
the optical cavity [16]. With a higher peak intensity, you
can create a higher resolution by creating a larger phase-
shift so the contrast is more visible (G. Jensen, 2015).
Just like the LPP the PLPP does not have a physical
phase plate so it has negligible electron loss due to the
phase plate. Besides, the phase plate does not deteriorate
over time as some physical phase plates do. [16].
V.3. Disadvantages
Due to the pulsed, and thus time-dependent, nature
of the laser beam the induced phase-shift is also pulsed.
This means that an additional step has to be taken into
account when processing the gathered data. This might
lead to additional time, needed for the data collection
and processing[15].
Because you want to keep the complexity of the PLPP
as low as possible a few constraints are introduced, an im-
portant one being that you can not have more electrons
in the beam than in a continuous beam like the LPP be-
cause otherwise the density would be higher and the elec-
trons would also start to interact with each other. This
means that fewer electrons will pass through/ past the
studied object per second so fewer data can be collected
per second [16]. A problem that increases the complexity
of the system is that the maximum possible phase-shift
induced in the electron beam only happens for a part of
the electron beam and not the whole beam. This might
be a cause for additional time needed in the data collec-
tion but it also might be a problem that can be solved
with further research [21].

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