Samenvatting Beeldvormende Technieken
2014
3D-vision
1a) Which physiological mechanisms play a role in 3D-vision? Explain how
this works.
There are four physiological mechanisms.
Accommodation
The lens of the eye changes in shape and thickness when
looking at objects at different distances. Tiny muscles in the
eye make this possible and with this actually change the shape
of the cornea. This also changes the refraction of light
travelling from air to eye, so that it forms a sharp image on the
retina. For accommodation only one eye is needed, but it works
together with convergence in order to perceive depth.
Convergence
Convergence is the angle made by the two
viewing axes of a pair of eyes. It is associated
with the eye muscles. The muscle tone can be
used to estimate the distance to an object. The
convergence of the axes of the eyes depends on
the distance to the object. The angle between the
axes of the eyes increases when an object is
very close and decreases when an object is
farther away.
Binocular differentiation
Binocular differentiation means that an object has different image locations on the
retina due to the distance of that object from the eyes. When the eyes focus on an
object, the optical axes form a parallactic angle. This angle depends on the distance of
this object from the eyes. The angle will decrease with increasing distance. So, depth
perception becomes more difficult with increasing distance. When you look at two of
the same objects at different distances, you can estimate relative distances by
comparing the different locations on the retina.
This mechanism is based on the natural horizontal separation of the eyes. This is why
you need 2 eyes to use this mechanism.
Motion parallax
Motion parallax: Using the movement of objects to estimate how
far objects are away in the scene you are looking at. Objects that
are closer to the eye appear to move faster through our field of
sight than objects farther away, even if the objects are moving at
the same speed. Moving the head of the one observing can cause
the same effect. This type of mechanism is important for
organisms that only have the use of one eye.
1a) What is chromostereopsys? How does it work?
Figure 1. The mechanism in
which the moving objects or
the movement of the viewers
head are used in estimating
the distance of objects in the
field of sight.
Chromostereopsys
Chromostereoscopy, is a method to see depth based on the visual
phenomenon of chromostereopsis. This system uses colors to
guess distance. The eye is not uniformly covered in rods and cones. The cones are
concentrated in an area called the fovea. As a result the visual axis isn’t the same as
the central axis of the eye. Light enters the eye and lands on the fovea at an angle.
Light of shorter wavelengths (blue) is refracted more than light of longer wavelengths
(red)(figure 2). That is way the colors appear to be at different positions (figure 3).
Figure 1. Light of shorter
wavelengths(blue) is refracted more
than light of longer wavelengths (red).
Figure 2. Blue objects
appear to be further away
than red objects.
1b) Which psychological mechanisms play a role in 3D-vision? Explain how
this works.
The six major psychological mechanisms that play a role are:
 Retinal image size
 Linear perspective
 Aerial perspective
 Overlapping
 Shade and shadows
 Texture gradient
These mechanisms are all based on experience and learning. In normal imaging these
cues are used together to get an image of the surroundings.
Retinal image size: the amount of space an object takes on the retina. If the retinal
image size of an object is bigger, it looks closer than an object with a smaller retinal
image size. The accuracy by which we can estimate the distance from the object depends
on our knowledge of the real size of the object.
Linear perspective: the gradual reduction of image size when the object goes further
away from you. This mechanism can be illustrated with the Ames room. If you look
through a hole into the room, it looks normal. But in reality the left corner is further
away than the right corner. When a person walks from the left to the right corner he
looks bigger by the time he comes closer to the right corner. An example of linear
perspective in normal situations is a road that disappears at the horizon. The road
seems to get less wide when it approaches the horizon.
Aerial perspective: the background of an image becomes fuzzy and blue. The bluer it
gets, the further away it looks. This is because the refraction of the light. So distant
objects look fuzzier. Atmospheric tinting means that when the whole image is fuzzier,
everything looks further away.
Overlapping: constant lines look closer than interfered lines. When a person overlaps
another person, you know the first person is closer than the second person.
Shade and shadows: there is not much difference between shade and shadow. Most of
the light comes from above and the image you get is based on this knowledge. For
instance, when a round circle is black on the upside and light under this, it appears as
hollow. When you turn the image upside down the circle looks like it comes to you. This
is dependent on your interpretation on how the light is supposed to fall.
Texture gradient: the closer an object gets, the more detail you’ll see. Further away, the
texture becomes smoother. This is called a texture gradient.
PET en SPECT
2a) What is tomography?
Tomografie is het samenstellen van een 3D-model door het stapelen van 2D-plakken. Deze
2D-beelden kunnen bijvoorbeeld worden verkregen door het gebruik van energie/radiatie
golven. Er zijn verschillende soorten tomografie, die grofweg kunnen worden verdeeld in
twee categorieën: structurele en functionele beeldvorming.
 Structureel: CT, MRI
 Functioneel: PET
2a) Explain the physical background of positron emission. What are they?
Bij een PET-scan gaat het om de radioactieve verval van de atomen. Een onstabiele kern
ondergaat radioactief verval om te kunnen stabiliseren, hierdoor krijgt hij de perfecte
proton-neutron verhouding. Een vorm van verval is met positronen. De positron is eigenlijk
de tweelingbroer van de elektron, het heeft dezelfde massa als de elektronen. Het enige
verschil is dat de positron positief geladen is. Bij de PET scan wordt gebruik gemaakt van het
verval. Er wordt een stof ingespoten die gelabeld is met een positron emitting radionuclide,
het heeft een instabiele kern. De radionuclide zal vervallen en hierbij komt een positron vrij.
De positron botst met een elektron en ondergaat een reactie, hierbij komen 2 fotonen vrij,
deze worden gedetecteerd.
2a) Which isotopes emit positrons? Why?
Er zijn een aantal grote isotopen die van nature positronen uitschieten. Deze isotopen
hebben van henzelf een te grote positieve lading en schieten daarom een positron uit om
stabiel te worden. Hieronder staan de 4 meest gebruikte isotopen en hun halveringstijd.
•
•
•
•
Carbon-11
Nitrogen-13
Oxygen-15
Fluorine-18
half-life: 20.334 min
half-life: 9.97 min
half-life: 122.24 min
half-life: 109.771 min
De halveringstijd is de tijd waarin de helft van de isotoop uiteen is gevallen.
De isotopen worden gebonden aan een tracer die metabool actief is. Dat wil zeggen dat de
tracer wordt opgenomen door de cel en wordt gebruikt in het metabolisme van de cel. De
tracer wordt samen met het isotoop intraveneus ingebracht. Voorbeelden van een tracer
zijn glucose en methionine.
De tracer glucose wordt bijvoorbeeld gebruikt bij het opsporen van kankercellen omdat deze
meer cellen meer glucose gebruiken in hun metabolisme dan normale cellen.
2a) What is a voxel and a pixel, what is a look-up table? Demonstrate the
effects of various look-up tables.
Een pixel is een twee dimensionaal vierkantje wat informatie geeft over een bepaald
gegeven. Wanneer er meerdere pixels op elkaar gestapeld worden vormt zich een drie
dimensionaal vierkantje wat een voxel genoemd wordt. Om de gegevens in een voxel te
kunnen verwerken is een look-up table(LUT) nodig. Meestal wordt er een lineaire look-up
table gebruikt. Maar om de kwaliteit van het beeld te verbeteren kan de look-up table
worden aangepast. Dit is te zien in de volgende afbeeldingen:
Width = 1153, level = 576
Width = 280, level = 860
Width = 320, level = 730
Width = 1, level = 470
2b) What is annihilation?
The technique of PET scanning is based on photons that are detected by registering the
resulting gamma rays. To obtain these photons, a certain process is needed, namely
annihilation. It all starts with a proton-rich isotope (PET radio-isotopes) with an
instable nucleus. This nucleus decays via positron emission, which causes a proton in
the nucleus to become a neutron. Positron and neutrinos are formed (neutrinos don’t
interact with matter, so they can be neglected in PET). You can imagine a positron as a
positive charged electron. The positron travels through the body where it interacts with
electrons. This process costs energy and when the energy is low enough, the positron
and an electron collide with each other. The two particles annihilate each other.
Annihilation is the conversion of the mass of an electron + a positron into light,
according to E=mc^2. So the process of annihilation produces two photons and these
protons travel in opposite directions (anti-parallel). The detection of the photons is
possible because of the presence of electromagnetic radiation. (Not visible by eye, the
wavelength is to short) Thus, by annihilation photons are released and can be measured
by a detector because of the presence of electromagnetic radiation.
2b) What is the mean free path?
The mean (not main) free path is the distance between the location at which a positron
emerges and the location where it has dissipated enough of its energy to be able to react
with an electron to yield annihilation. Easier to imagine as the distance a positron
travels before annihilation, it’s about 1 mm.
2b) How is a PET camera composed?
A PET camera is composed of detectors that are coupled to photomultiplier tubes. These
photomultiplier tubes are connected to a computer system, which creates multicoloured two- or three-dimensional images. When the detector registers an incident
gamma photon produced by annihilation it generates a timed pulse. These pulses are
combined in coincidence circuitry. The pulses are considered to be coincident if they fall
within a short time-window. So, signals that are detected at the same time are registered
as coincidence events; the signals come from the same annihilation.
2b) What is the spatial resolution of PET?
The main free path of positron emission results in a worse spatial resolution, because
the site of emission differs from the site of annihilation. The positrons travel a while
before annihilation. Thereby the detector measures not the exact position of the nucleus
(site of emission), but it measures the position of annihilation. Also the size of the
detector influences the spatial resolution.
2b) What is the temporal resolution?
The temporal resolution refers to the accuracy of the measurement time wise. The
temporal resolution of PET is very low in reference to other types of measurements.
This is due to the fact that PET makes use of nucleus delay, which takes several minutes
to set in. This causes the resolution to be tens of seconds to minutes, which severely
affects the amount of measurements one can do.
2b) Why is PET still of interest?
PET scans, unlike CT or MRI scans, can measure cellular-level metabolic changes
occurring in an organ or tissue. This results in an advantage for PET over other
technologies to detect whether or not a lesion in the body is benign or malignant. This
can sometimes eliminate the need for surgical biopsies if PET scans rule out malignance.
This advantage also helps detecting diseases by recognizing disease processes at the
beginning with only functional changes, whereas CT or MRI won’t detect any changes
until they cause structural changes in the organ or tissue.
2b) What is SPECT?
SPECT is an abbreviation of Single-Photon Emission Computed Tomography. SPECT is
an imaging technique that shows how blood flows to tissues and organs. While SPECT
shows a lot of similarities to PET, SPECT is different in the use of its radioactive isotope.
SPECT uses a radioactive isotope, which directly emits a gamma ray upon radioactive
decay. This results in one ray instead of two, as with PET, which causes a lower spatial
resolution in SPECT images as opposed to PET. However, partly due to more obtainable
and more sustainable isotopes used in SPECT, this technique is much less expensive
than PET.
MRI and fMRI
3a) Explain how protons behave in a magnetic field.
Protons are positively charged hydrogen nuclei with a small mass and a North and South Pole. A
proton spins around its own axis randomly and therefore creates a magnetic field. In the absence
of an external magnetic field, the proton will not point in a specific direction but will just tumble
randomly (figure 1). When placed in a magnetic field of an external source, the proton will align
or anti-align with the direction of the field in a ratio of approximately 1:1 (figure 2). There should
be noted that the protons never align exactly with or against the magnetic field; there is always a
small divergence between the axis of the proton and the axis of the field. The protons constantly
switch orientation and when there are enough protons present, there will be a slight excess of
protons in the aligned orientation creating a magnetic vector oriented along the axis of the
external field. This is because the energy-state in which the aligned protons are, is lower and
therefore energetic more favourable than the energy-state in which the anti-aligned protons are;
these differences get larger when the external magnetic force increases (figure 3).
3a) Subsequently the protons are exposed to electromagnetic radiation (a
radio wave). What can happen to the proton? Under what conditions does this
occur?
When an electromagnetic radio wave (RF) with a frequency equal
to the precession frequency (the Larmor frequency) is applied,
protons will absorb a specific amount of energy. As a consequence
one single proton will jump to a higher energy state. Therefore the
magnetization vector (Mø), which is the total magnetic field of the
excess protons, will spiral down to the XY plane (Figure 4). Only
when the frequency of the radio wave is exactly equal to the
precession frequency, the magnetization vector will spiral down.
Figure 4
3a) What is the Larmor frequency?
When placed in a magnetic field, the protons align either parallel
or antiparallel (view above). The protons spin around the
straight line of the magnetic field which causes a difference
between the axis of the proton and the axis of the magnetic field.
This is called precession (figure 5). The Larmor frequency is the
frequency of the precession of a charged particle when its
motion comes under the influence of an applied magnetic field
and a central force. The Larmor frequentie can be calculated by
the so called Larmor equation:
Where ω is the Larmor frequency in MHz, γ is the gyromagnetic ratio in MHz/Tesla and B is the
strength of the static magnetic field in Tesla. The gyromagnetic ratio is the ratio between the
magnetic moment and the angular momentum (the amount of rotation an object has).
3a) What happens if the radio wave is turned off?
When the radiofrequency source is switched off, the
magnetic vector returns to its resting state and this
causes a signal (also a radio wave) to be emitted. This
signal is used to create the MR images. The hydrogen
protons give back their absorbed energy and return
slowly to their natural alignment. The emitted energy
can be measured. With this data it is possible to
determine the density of the protons. When they do
this, they give off a signal that the coils pick up an send
to the computer system (step 4 and 5; figure 6).
Receiver coils are used around the body part in
question to act as aerials to improve the detection of
the emitted signal. The intensity of the received signal
is then plotted on a grey scale and cross sectional
images are built up.
Once the RF transmitter is turned off three things begin to happen
simultaneously.
1. The absorbed RF energy is retransmitted;
2. The excited spins begin to return to the original Mz orientation;
3. Initially in phase, the excited protons begin to dephase.
1. Once Mz (a magnetization vector) has been tipped away from the Z axis, the vector
will continue to process around the external Bø field at the resonance frequency ωø. A
rotating magnetic field produces electromagnetic radiation. Since ωø is in the radio
frequency portion of the electromagnetic spectrum the rotating vector is said to give
off RF waves. The absorbed RF energy is now being retransmitted, thereby producing
the NMR signal.
2. The process of giving off RF energy occurs as the spins go from a
high-energy state to a low-energy state, realigning with Bø. The RF
emission is the net result of the Z component (Mz) of the
magnetization recovering to Mø. Not all of the energy given off is
detectable as an RF pulse. Some of the energy goes to heating up the
surrounding tissue, referred to as the lattice. In a global, or rather,
universal sense, this system can be divided into the spins, and the rest
of the universe, or a very large lattice. This type of spin-lattice
interaction is the result of the excited system returning to thermal
equilibrium.
3. The time course whereby the system returns to thermal equilibrium, or Mz goes to Mø, is
mathematically described by an exponential curve. This recovery rate is characterized by the
time constant T1, which is unique to every tissue. As will be discussed in detail later, this
uniqueness in Mz recovery rates is what enables MRI to differentiate between different types of
tissue. At a time t=T1 after the excitation pulse, 63.2% of the magnetization has recovered
alignment with Bø.
3b) Now explain Magnetic Resonance Imaging.
Every proton has its own spinning, named precession, which causes an
electric field. This electric field gives the protons a direction. The MRI-scanner
contains two big magnets. These magnets create a magnetic field in which
the protons align with the magnetic field or align against the magnetic field. So
the protons spin again, but this is a different type of spinning. A little more
protons align with the magnetic field than against the magnetic field. The MRI
scanner sends out random radio frequencies because the radio wave
frequency has to be exact the same as the precession of the protons for the
protons to absorb the energy of the radio waves. The absorption causes a
turn of the protons by 90-180 degrees. The longer the radio waves are
applied, the more the protons get tilted. When the radio waves stop, the
protons emit the energy that was absorbed and the
protons return to their original state. The signals that
return are different in each kind of tissue. The emitted
energy can then be measured. In this way the density
of the protons can be determined and an image can
be created.
3b) What is the purpose of the gradient of the magnetic field?
If you have a homogenous magnetic field, the strength of the field is
everywhere the same. This has as result that every proton that has the same
precession will absorb the same radio frequency and will emit out the same
signal. In this way you can’t locate where the signals come from.
An inhomogenous magnetic field differs in strength of the field. The gradient
results in differences in precession of the protons. Because of this, the
protons absorb also different radio frequencies and emit different signals. In
this way different signals return to the MRI and then it is possible to locate
where the signals came from.
3b) What is fMRI?
Functional MRI is used for neuroimaging. Hemodynamic responses on neural
activity during tasks are measured. The fMRI is a comparison of two MRI
scans made during a task and during rest. In these scans the consumption of
oxygen is measured, also known as the BOLD technique.
3b) What is the BOLD technique?
BOLD stands for Blood-oxygen-level dependent signals. The BOLD technique
is used to determine where the neural activity is. Oxygenated hemoglobin has
a lower magnetic field than deoxygenated hemoglobin. This is because of the
own magnetic field Fe had when it hasn’t bound oxygen.
3b) What is angioMRI?
AngioMRI makes use of MRI scans to make images of the arteries, to
investigate if there are any stenosis or aneurysms in the vessels or arteries.
There are two different methods, Flow dependent angiography and Flow
independent angiography. Flow dependent angiography uses signals from
moving blood vs static tissue. Flow independent angiography uses contrast
agents like iodine, which makes the blood vessels brown.
X-ray tomography
4a) What are X-rays? How are they generated.
X-ray computed technology is a technology based on tomography that uses x-rays to
produce virtual slices (layers) that produce an image of specific areas of the scanned
object. X-ray is electromagnetic radiation and uses a wavelength between 0.01 and
10 nanometres. There are different kinds of X-rays that are used for different kinds of
tissues. X-ray was discovered by Wilhelm Röntgen in 1895. Tomography means
imaging by sections. In computed tomography, or CT, X-rays are used to image.
X-ray computed tomography is used in X-ray Crystallography, Mammography,
Medical CT and Airport Security. All use different wavelengths and photon energy’s.
The wavelength used is in between visible light and gamma rays. The different
wavelengths will show different body tissues. For example, lower wavelengths are
used for mammography, higher wavelengths are used in detecting machines in
airports.
4a) What equipment is needed?
X-rays are created by a difference in voltage between the anode and the cathode.
The electrons want to move from the cathode (which is the negative part) and the
anode (the positive part). This causes a electron flow towards the anode, which is
called an electron beam. The electrons bump out an electron from the anode. When
the same (or another) electron moves back, it will send out photons. These are the xrays. Every ray that hits the screen, will turn black. These rays are not absorbed by
the body, for instance.
The anode’s material is very important, because different anodes will get different
graphs. There is an oil bath in the devise, which is used to cool down the whole
devise. The motor turns around so the generated heat is distributed. A vacuum tube
is used so water or air won’t disrupt the image. The filter in the X-ray machine is
necessary for creating the desired wavelength.
In brake radiation, a passing electron is lowered in energy by being slowed down by
the atom core and hereby energy is released as radiation waves. In collision a
colliding electron bumps an electron from a higher orbit to a lower orbit. This creates
radiation waves as well.
4b) Describe recent progress of XCT.
The first scanner was called a single-slice axial CT-scanner. It had a single emitter
and a single detector. It made single images over a single axis. A disadvantage was
that it took 30 minutes to make a scan. Another disadvantage is that the patient was
exposed to the radiation for 30 minutes. The last disadvantage is that you had to turn
the machine to get a full image of the body.
The second generation had a fan shape beam and had multiple detectors. It had still a
single axis, so you still had to adjust it. An advantage was that it only took 10 minutes
to make a scan.
Slip-ring technology: there are moveable emitters and detectors in a ring, so you get a
helical view. This creates more information. There is also overlapping between slices.
The distance between the slices is a pitch. When there is a low pitch, there is a higher
overlap and a longer scanning time. It takes longer to scan the whole distance you
need. With a high pitch you can scan a longer area of the body in a shorter time.
Nowadays there is a multi-slice CT-scan. There are 4+ detectors and multiple rays.
There is still a helical view and more overlapping ribbons. This increases a better
view. The scan is quicker and you can scan a larger part of the body. The scan is
therefore safer, because the amount of radiation that the patient is exposed to is much
lower. There is also a possibility of 3D images.
4b) Explain the principles of the recently introduced dark-field method.
The dark-field method: improving the contrast of a picture by only using scattered
light. There are two gratings: the second one (G2) is placed behind the first one (G1),
so that the holes of the first one are blocked. This creates a dark field, because no light
can pass the second grating. When you put an object in front of the first grating, it
creates scattering. Light gets through the two gratings, so that it can be detected. The
background is not scattered. When light passes the first grating, it will also result in
diffraction, which makes extra noise. Diffraction patterns repeat itself, after a certain
distance. This is the Talbot effect. If you place the second grating at that distance, the
diffracted rays will be blocked. Furthermore, the use of homogeneous light reduces
diffraction. This is created by G0. The advantage of the method is that you can create
a higher contrast. The disadvantage of the method is that the patient is exposed to a
high dose of radiation
4b) Give an example of the improvement of image quality.
On the picture you can see an image of a chicken wing. The
left image is a conventional transmission image and the right
image a dark-field one. Scattering of the chicken bones
creates a strong signal in the dark-field image. Even though
you can already see the bones in the transmission image, the
dark-field image provides an enhanced contrast. This enhanced contrast can help
doctors see micro fractures in bones, which they wouldn’t be able to see on a
conventional transmission. The image on the right however requires a high dose of
radiation. This means it might not be as good as it seems.
Electron microscopy
5a) Explain what electron microscopy is.
Electron microscopy is a technique that uses a beam of electrons and an
electromagnetic field to visualize a specific object. In contrast, a normal light
microscope uses beams of photons and glass lenses.
Functioning of the microscope consists of a few phases. First an electron gun fires
electrons at an object. This beam is focused by electromagnetic fields. The electrons
interact with the object, but not every part of the object interacts the same way with
the electrons, because every substance differs in the admittance of electrons. After
transmission, the electrons are captured by a fluorescent screen. The number of
captured electrons gives an indication of the density of the substance. This is what
creates the image.
5a) What is the wavelength of an electron?
The wavelength of an electron depends on its speed. This is determined by the voltage
(Va) that is used for the acceleration of an electron. The formula to calculate the
ℎ
wavelength of the electrons is 𝜆 =
(Planck’s law).
√(2𝑚𝑒𝑉𝑎 )
A commonly used acceleration voltage is 100 kV. At Va = 100 kV, electrons have a
wavelength of 0,004 nm.
5a) What is the spatial resolution of an electron microscope?
The spatial resolution of an EM is extraordinary high in comparison to a light
microscope (0,1 nm versus 1 μm respectively). This is due to the fact that the
wavelength of electrons is a lot smaller than the wavelength of visible light.
5a) What are primary electrons, secondary electrons, backscattered
electrons, Auger-electrons?
- Primary electrons: The electrons that are shot out of the electron gun.
- Secondary electrons: When a primary electron pushes an electron out of
the orbital of an atom, this pushed-out electron becomes a secondary
electron.
- Auger electrons: A primary electron pushes an electron out of the core
orbital of an atom. A higher energy electron from an outer orbital will
take its place. The energy that is generated by this process will accelerate
another outer orbital electron. This electron is called the Auger electron.
- Backscattered electrons: Electrons from the gun interact with the object in
a way that they will scatter back towards the gun.
5a) What is the difference between elastic and non-elastic scattering?
- Elastic scattering: An interaction between the electron and the specimen
that doesn’t cause energy to be transferred from the electron to the
specimen.
- Inelastic scattering: An interaction between the electron and the specimen
that causes part of the energy or all of the energy of the electron to be
transferred to the specimen.
Ant
Pollen grains
5b) What are the essential differences between Scanning Electron
Microscopy and Transmission Electron Microscopy? What causes the
appearance of contrasts?
Scanning electron microscopy:
Electrons are used to get an image of your specimen. The specimen has to be in a
vacuum, because molecules in air or water will cause electrons to scatter. The whole
specimen gets scanned in a raster pattern and from there you can visualize an image.
Primary electrons come from an electron gun, an anode lets those electrons accelerate.
Electromagnetic fields and lenses are used to focus the beam in a straight line on the
specimen. A condenser lens is used to create a small spot of electrons, which scans the
specimen. The size of this spot determines
TEM
the resolution. SEM detects backscattered SEM
electrons, secondary electrons and x-rays.
Low energy electrons
High energy electrons
Backscattered electrons are reflected after
collision with the specimen, whereas
Scanning beam
Fixed beam
secondary electrons are emitted by the
molecules of the specimen. The amount of Image by: sec. e- and
Image by: primary eemitted secondary electrons and their
back-scattered evelocity depends on the height and the
Resolution max 1 nm
Resolution max 0,1 nm
slope of the surface. A tilted surface emits
more electrons than a flat surface. When
2D (‘3D’) image: surface 2D image
more electrons are emitted, the signal will
be brighter. X-rays aren’t used for imaging,
Contrast: back-scattered
Contrast: scattering and
but give characteristic chemical
einterference
information about the emitting atoms.
2D images (look like 3D) are produced of
Object between detector
Object between condenser
surfaces. Contrast caused by: difference in
and objective
and objective
electron scattering by different atoms.
Transmission electron microscopy:
Transmission Electron Microscopy works with thin slices of specimen (about 100 nm).
These can be treated with heavy metals. These slices have to be thin, because many
electrons should be able to pass through the specimen. The electrons that pass through
reach the base plate. There they react with a material that is able to absorb the electrons
and convert it into light. The pattern that has been formed can be seen on the plate. The
different structures in the specimen cause the contrast. When electrons hit atoms with a
large nucleus, they rebound. Electrons can be repulsed/absorbed in thick/dense areas.
The electrons are likely to pass through thinner or less concentrated material. The
traversing electrons eventually reach the scintillator plate that converts their energy into
light flashes. This differs for each part of the specimen by foretold reasons. The image
that is produced greatly corresponds with the differences in reflection and transmission of
the specimen. Electromagnetic lenses cause an enlarged projection because they
enlarge the amount of the exiting electrons. In order to get a higher contrast or study a
specific element, heavy metals are used to stain the specimen. These heavy metals
rebound more electrons and therefore the contrast will be higher. There are two types of
scattering that affect image formation: elastic (interact with nuclei and don’t change
speed/energy but direction) and inelastic (interact with electrons and lose energy/speed
and change direction) scattering.
2D images are produced of mainly cellular or other molecular structures.
Contrast caused by: elastic and inelastic scattering and interference.
5b) What is Cryo Electron Microscopy? How is it done, for what purpose?
Cryo-electron microscopy is a form of transmission electron microscopy that uses rapidly
frozen specimen. The specimen is frozen by using liquid nitrogen. After freezing it, the
sample is surrounded by amorphous ice and no ice crystals are present. After freezing,
the specimen is placed in a cryo-chamber. From outside a knife can be used to fracture
the specimen. Finally a thin layer of gold-palladium is ‘sputtered’ on the material, to
improve the conductance of the electrons. By freezing the specimen rapidly, vulnerable
structures are preserved. The specimen can also be fractured without damaging other
structures. Other advantages are that there is less radiation damage and dehydration
does not occur. This technique is mostly used to visualize vulnerable biological
structures, because it preserves their native structure.
2D image of very vulnerable biological structures.
Optical imaging and LASER technology
6a) Explain the principles and transmission pathways of a normal light
microscope.
Microscope and its parts
The microscope lamp is of low voltage but high intensity to
generate a lot of light. The light goes through the condenser,
which helps to evenly illuminate the sample. This is essential at
higher magnifications to give detail and sharpness to the image.
The diaphragm helps to control the solid angle of light that comes
from the condenser.
The objective provides the details we see in the final image. The
light then travels to the eyepiece. The image is further magnified
here.
The cornea and eye-lens are the final part of the transmission
pathway. The lenses of persons with normal vision will be
relaxed if the person looks into the microscope.
Path of light through the microscope
The Köhler setup
Köhler illumination is a method of specimen illumination used for transmitted en reflected
light optical microscopy. Köhler illumination acts to generate an extremely even illumination
of the sample and ensures that an image of the illumination source is not visible in the
resulting image. Important is to distinguish clearly between the illumination of the object and
the adjustment of the microscope. It must be mentioned that the diaphragm of the lamp (and
not the lamp philament itself) is focussed on the object. The objective lens creates an image
of the object, which is viewed through the eyepiece in such a way that no accomodation of the
lens of the eye is needed.
Resolving power of the microscope
The resolving power of the microscope depends on the limit of resolution caused by the
wavelength of light and the numerical aperture of the objective.
The gap where light can pass through is
indicated in the figure with ‘b’. This is larger
than the wavelength of the light passing
through. This creates interference patterns.
Interference results from differences in
optical path between the rays that are emitted
at the level of the diaphragm. If the
difference in path between two rays equals
half a wavelength, the resulting intensity is zero, and if the difference equals the wavelength,
the intensity is highest. Careful consideration of optical paths leads to an explanation of the
diffraction pattern: If the difference in path between a ray from the center of the diaphragm
and a ray at the rim of the diaphragm equals half a wavelength, than for every ray in one half
of the diaphragm a ray exists in the other half that has half a wavelength difference in path.
Therefore every ray is extinguished by another one, and darkness is the result. In this way the
image of one point of the sample exists of a circular spot (called Airy disc) and concentric
rings around it. Another nearby point of the sample also generates an airy disc with rings. As
soon as the points are so close together that the airy discs partially overlap, the image appears
as one blob of light, not two points. This is why diffraction limits resolution
The ability of the objective to accept diffracted rays is dependent on the refractive index of
the media.
In other words this means that not all of the detail of the specimen can be accepted by the
objective, some will be captured and will not be visible.
6a) What is the difference between magnification and spatial resolution?
Magnification:
The Numerical Aperture (N.A.) defines the limit of detail which can be resolved by the lens.
It can be calculated with the following formula:
N.A. = R.I. sin Ø
R.I. is the refractive index of the medium.
Ø is the angle the ray makes with the optical axis.
Magnification determines what degree of enlargement this detail is presented to the eye.
Resolution:
Resolution is about details whether magnification is about the degree of enlargement this
detail is presented to the eye. Resolution is limited by wavelength of light and the angle of
acceptance of rays by the objective. So increasing the resolution will increase the details, but
increasing the magnification will not lead to an increase in detail.
6b) Explain the principles of dark field microscopy, provide
examples.
Dark field microscopy is a bit like bright field
microscopy but it uses slightly different principles.
A special disk (the Darkfield stop) blocks some light
from the light source leaving only an outer ring of
light that passes the condenser lens which focuses
the light onto the specimen. Some of the light gets
scattered by the specimen, this light is captured by
the objective lens. The rest of the light, which isn't
scattered, is blocked by the objective aperture. The
scattered light produces an image on the retina of
the eye while the rest of the light is blocked,
creating the appearance of an illuminated specimen
on a dark background.
Dark field microscopy is mostly used for small light-diffracting specimens like
bacteria, isolated organelles and single celled organisms.
6b) What is a LASER?
LASER stands for Light Amplification by Stimulated Emission of Radiation. It is
based on stimulated emission of electromagnetic radiation and the energy states of
electrons.
6b) How does it work?
There are 3 mechanisms where photons interact with
atoms:
1. Absorption: No photons are released when a
photon interacts with an atom
2. Spontaneous Emission: There is a random
spontaneous release of photons.
3. Stimulated Emission: Two photons are released, in
the same direction and phase, when a photon
interacts with an atom.
LASER uses stimulated emission. The mechanism used in
LASER is called population inversion.
The principle behind population inversion is to keep
electrons in a higher energy state. It can only occur if one
of the energy levels has a relatively long half-life. If this is
the case, applied light can create a surplus of electrons in
that energy state. It turns out that photons that have exactly the same energy as
the electrons of the inverted population need to move to their lower energy level can
trigger the electron to move. The transition will create an additional photon of the
same wavelength, direction and phase. If there are 4 energy levels (E1 to E4), you
want to keep the electrons in an energy state between E2-E3. If an electron goes into
E2 it will fall to the ground energy level causing spontaneous emission, which you
don't want. By keeping the electrons in the stimulated emission zone between E2
and E3, you can double the photons everytime a photon interacts with an atom.
LASER also makes use of something called a resonator. A resonator is basically two
mirrors aligned perfectly parallel to each other. These mirrors ''bounce'' the photons
back and forth creating a wave. These waves have a certain frequency which can
exit a tiny hole which creates a laserbeam. The photons that don’t exit the hole keep
on bouncing back and forth between the mirrors, so they have a higher chance of
stimulating other electrons to go to a lower energy level. This way the beam gets
very intense.
6b) What is it for?
LASER can be used for all sorts of things: medically, commercially, for the military
and microscopy.
Think of the laser pointers that are used in lectures, laser cutters or confocal laser
scanning microscopy. These are just a few examples of the huge amount of things
LASER can be used for.
Phase-contrast and Interference microscopy
6c) What is phase-contrast microscopy?
Phase contrast microscopy was invented in 1934 by Dutch physicist Frits
(Frederik) Zernike (1888 - 1966), who was awarded the only Groninger Nobel
Price for it. The main aspect of this invention is to convert phase differences
into amplitude variations that can easily be detected. For instance, phase
contrast (PC) microscopy can be used to produce high- contrast images of
transparent specimens, such as living epithelial cells. It is an interference
technique that requires at least partially coherent light to illuminate the
specimen (more precisely, partial, longitudinal coherence is required to make
PC work).
6c) How does it work? When
is it used?
The basic principle to make
phase changes visible in
phase contrast microscopy
is to separate the
illuminating background light
from the specimen scattered
light, which make up the
foreground details, and to
manipulate these differently.
The light (green) that passes the condenser annulus is focused on the
specimen by the condenser, the bundle of light is now a hollow cone, thanks
to the geometry of the annulus.
Some of the illuminating light is scattered by the specimen (yellow). The
remaining light is unaffected by the specimen and form the background light
(red). When observing unstained biological specimen, the scattered light is
weak and typically phase shifted by -90° — relative to the background light.
This leads to that the foreground (blue vector) and the background (red
vector) nearly have the same intensity, resulting in a low image contrast (a).
In a phase contrast microscope, the image contrast is improved in two steps.
The background light is phase shifted -90° by passing it through a phase shift
ring. This is a ring which has a cove in the disc. This cove is less thick so the
phase shift comes from the difference in index of refraction (or: speed of light
in a medium.) This phase shift eliminates the phase difference between the
background and the scattered, leading to an increased intensity difference
between foreground and background (b). Some of the scattered light will be
phase shifted and dimmed by the rings. However, the background light is
affected to a much greater extent, which creates the phase contrast effect.
The above describes negative phase contrast. In its positive form, the
background light is phase shifted by +90° instead. The background light thus
will be 180° out of phase relative to the scattered light. This results in that the
scattered light will be subtracted from the background light in (b) to form an
image where the foreground is darker than the background, as shown in the
figure. Two things happen:
Refraction


Different index of refraction, different phases and resulting ray will be
refracted.
Refraction large enough, will not pass through the phase-ring in the
phase-plate
Diffraction




Light interacts with atom, induces vibrations in certain electrons
Vibrating electrons emit light
Image appears 90 degrees out of phase, cancelling each other out
Creates contrast
6d) What is differential interference contrast (DIC) microscopy?
Differential interference contrast microscopy (DIC) is an optical microscopy
illumination technique used to enhance the contrast in unstained, transparent
samples. It works on the principle of interferometry, which is a family of techniques
in which waves, usually electromagnetic, are superimposed in order to extract
information about the waves. So DIC takes advantage of differences in the light
refraction by different parts of living cells and transparent specimens and allows
them to become visible during microscopic evaluation. DIC gives a greater depth of
focus allowing thicker specimens to be observed under higher magnifications.
6d) How does DIC work?
To create contrast polarised light is needed. A differential interference microscopy
can create polarised light. This polarised light (45°) travels through a Wollaston
prism, causing the beam to be separated into two rays: a sampling ray and a
reference ray. These two rays will travel through a condenser to concentrate the
light, followed by traveling through the sampling object. The two rays will pass
through the sampling object in adjacent points, causing the sampling object to be
illuminated by two light sources; one with a polarisation of 0° and one of 90°.
The area’s in the sampling object will have different refractive index or thickness,
allowing the rays to get different optic paths. This causing a phase shift of one ray
compared to another.
After passing the sampling object the rays will
pass through an objective lens and another
time through a Wollaston prism. The last
Wollaston prism will combine the two rays to
one with a polarisation of 45°. The result is
blocked by the 2nd polarisation filter (135°).
Only if the two beams through the sample
experience different optical path length will
the combined beam have a polarisation
different from 45° and partially pass through
the 2nd polarisation filter. The merging of the
rays leads to brightening, darkening and
interference of the image. See figure 1 for the
schematic set up of a DIC.
6d) When is DIC used?
Differential interference contrast microscopy (DIC) has strong advantages in uses
involving live and unstained biological specimen. Its resolution and clarity in
conditions such as this are unrivaled among standard optical microscopy techniques.
It enhances the contrast in unstained, transparent samples.
The main limitation of DIC is its requirement for a transparent specimen of fairly
similar refractive index to its surroundings. DIC is unsuitable (in biology) for thick
specimen, such as tissue slices, and highly pigmented cells. DIC is also unsuitable for
most non biological uses because of its dependence on polarisation, which many
physical specimen would affect.
So in short the advantages are the high resolution, good contrast and it requires no
staining. The disadvantages of this technique are that it needs thin transparent
samples, it appears in 3-D and gives no effect on birefringent specimens because of
polarization.
6d) Provide examples
DIC is often compared with phase contrast microscopy, they just show different
details of your specimen. Some examples of the difference in details between these
two techniques is given figures 2 and 3.
links: DIC, rechts: fase contrast microscoop
boven: DIC. Onder: fase contrast
EEG, MEG, SQUID
7a) Explain the origins of EEG signals. How can EEG signals be used to
generate 3D images of the underlying current sources. Provide examples.
Bioelectricity
Neurons
Electroencephalography is a method to record the spontaneous rhythmic electrical activity of
a brain through electrodes attached to the scalp. Neurons in the brain are electrically charged,
thereby maintaining the brain’s electrical charge or also called bioelectricity. This electric
charge is maintained by billions of neurons. Cortical neuron activity generates electrical
signals that change in the 10 to 100 millisecond range. Due to this, EEG has an extremely
high temporal resolution.
Voltage generation
Membrane transport proteins pump ions across the neuron membrane and are constantly
exchanging ions with the extracellular milieu. When many ions are pushed out of many
neurons at the same time, it creates a wave of ions pushed out of neighbouring neurons. This
wave of ions can reach the electrodes on the scalp and push or pull electrons on the metal of
the electrodes. Metal conducts the push and pull of electrons easily. The difference in push or
pull voltages is measured between two electrodes. The voltages over time
are measured by a voltmeter and give the EEG.
Synchronicity and parallel orientation
An individual neuron generates an electric potential that is far too small to
be picked up by EEG. Therefore, EEG activity always represents the
synchronous activity of thousands or millions of neurons that are orientated
parallel relative to each other. If the cells do not have similar spatial
orientation, their ions do not align and create waves to be detected.
Pyramidal neurons of the cortex are well-aligned and fire together, thereby
producing the most EEG signal. Activity from sources deeper inside the
brain is more difficult to detect than currents near the skull.
EEG wave formation
EEG
The detected signals are transmitted to an EEG system
composed of amplifiers, filters, and paper chart or computer
monitor. Each electrode is connected to one input of differential
amplifier with one amplifier per pair of electrodes. A reference
electrode is connected to the other input of each amplifier.
These amplifiers amplify the voltage between the active
electrode and the reference. A channel represents voltage signal
and is depicted as a waveform in the EEG. The more channels are used, the higher the
accuracy of the EEG.
Standard set-up
The recording is obtained by placing electrodes on the scalp with a conductive gel or paste.
Two anatomical landmarks are used to position the electrodes: the nasion and the inion,
which are the area between the eyes, just above the bridge of the nose and the lowest point of
the skull from the back of the head respectively. In the standard set-up according to the lead
system, 19 electrodes are placed uniformly along the scalp. In addition, one or two reference
electrodes are required.
Wave forms
An EEG signal is depicted in a number of graphs, in which the voltage is shown on the
vertical axis and the time on the horizontal axis. The shape of the graph gives information
about the state of the brain functioning, for example sleeping, excited or relaxed state. Scalp
EEG activity shows oscillation at various frequencies. Several of these oscillations have
characteristic frequency ranges, spatial distributions and are associated with different state of
brain functioning, like waking and the various sleep stages.
Artifacts
Electrical signals that originate from non-cerebral sources
are called artifacts, which are almost always present in the
EEG data. The amplitude of artifacts can be quite large
relative to the size of amplitude of the cortical signals of
interest. Some of the most common types of biological
artifacts include eye blinks, eye movements and eye
muscle activity.
Event-related potential
Event-related potential are commonly used in experiments in the field of cognitive
neuroscience. A subject is repeatedly exposed to a certain type of stimulus, while at the same
time the EEG is created. An average is calculated from the EEG signals, yielding a mean
brain response to the stimulus. In other words, it gives a depiction of only the brain activity
induced by the stimulus. This way, the response of the brain to different type of stimuli can be
compared.
Creating a 3D image
EEG data can be used to evaluate EEG characteristics. This can be
done by spectral analysis and 3D imaging. Spectral analysis
focuses on the oscillations that can be observed in EEG signals. It
provides more detail concerning frequency over time. EEG
topography allows mapping of electrical activity across the surface
of the brain. It visualizes frequency over time corresponding to a
certain region of the brain, providing spatial information. Spectral
analysis and topographical mapping can be used together to create
a 3D image of the brain activity.
Source localization
EEG has a low spatial resolution, because a measured electrical signal can originate from
different sources in the brain. In fact, the same signal can be produced by many different
possible sources. Therefore, source localization is stated as a problem and a solution is
required. It is an inverse problem, as EEG data is used to find the sources. Reverse modelling
is used for the reconstruction of the source and aims to give an estimation of the location of
the source. The accuracy of this method is never 100%, because
more than one distribution of the current sources lead to
the same EEG. Hence, more knowledge (for instance
about conductivity of the brain) is required to distinguish
between the possible solutions of the analysis.
7b) What is MEG?
MEG is a functional imaging technique that
measures magnetic fields generated by neuronal
activity. More specifically, it measures the
magnetic fields created by post-synaptic
potentials. In this way MEG has a higher temporal
resolution than other
imaging techniques such
as fMRI and PET, which measure fuel
consumption. Neuromagnetism is the term that
is being used for the magnetic fields that are
generated by neuronal activity. Neuromagnetism
is created simply because electricity flows through
Depiction of the
application of the
the neurons. By using the right-hand rule it can be right-hand rule to a neuron. The white
determined what the direction of the magnetic
arrows indicate the direction of the
field is (see figure 1).
electric current whilst the red arrows
depict the direction of the electromagnetic field.
7b) Describe the required equipment for MEG.
The magnetic field of the neurons is very small. For
this reason special equipment is
needed in order to
be able to measure them. First of all, it is it necessary
for MEG
to be executed in a so-called magnetic
shielding room (MSR). The walls of this room
are covered with alternating layers of aluminum and alloy. In addition, active
shielding is used. The active shielding system senses incoming signals from outside
the room, and produces signals of compensating voltages to annul as much of the
incoming signal as possible. In theory, MEG can be more precise than EEG, because
the skull will less distort the magnetic signal than the electric signal that is being
measured with EEG.
7b) What is SQUID?
SQUID stands for superconductive quantum interference device. In MEG, many
SQUIDs are being used in order to take measurements. SQUIDS are able to detect
very subtle magnetic fields and are thus convenient for measuring the small electromagnetic fields created by neurons (see figure 2). Two main principles derived from
quantum physics are of importance for SQUID: 1) superconductivity and 2) electron
tunnelling. Both of these will be discussed in the section below.
This image shows a rough
division between the
strengths of electromagnetic fields of
different objects. It is clear
that femtotesla under
which human brain
activity falls is a rather
small amount of
electromagnetism
compared to other objects.
Superconductivity:
Superconductivity is the process in which the cooling down of
some particular metals will lead to an electrical resistance of zero. The metal has to
reach a ‘critical temperature’ for this to be able to happen. When the resistance of
the metals has reached a value of zero, they are said to be in a superconductive
state. When these metals are in this superconductive state, they try to stay there. In
order to be able to do this, the metals create a so-called ‘screening current’ or
Iscreening. This current causes there to be both a current going down and a current
going up. The result of this is that both currents will cancel each other out. This is
otherwise known as the Meissner effect. Ultimately this means that there is zero
resistance.
Electron tunneling:
Quantum physics is a probabilistic theory. It states that we can
never exactly know where an object is, but merely where it is most likely to be. This
can thus also be said for electrons. Whilst classical physics assumes that electrons
revolve around the nuclei of atoms in circles, quantum physics states that electrons
make a probability wave around the nuclei of atoms and that they do not orbit the
nucleus in this classical manner. In 1962, Brian Josephson demonstrated the
existence and characteristics of the so-called “tunnelling-effect”. This effect can be
produced between two superconductive materials separated by a thin insulating
layer. This is called a Josephson junction and is also used in SQUID. The Josephson
junction enables the flow of electrons, even in the absence of an external voltage. In
SQUID, the electron current flow caused by the Josephson junctions flows around
the ring. The magnetic field that is measured by the ring will change this electron
flow and this will affect the overall current flow across the SQUID. The exact
mechanisms of the SQUID are described further below.
7b) Describe therequired equipment for SQUID.
A SQUID is created out of a superconductive ring containing liquid helium (for the
cooling down) that has two barriers built-in. These barriers are known as Josephsons
junctions and are non- superconductive. In the absence of an external magnetic field
the current that will run through the ring will be known as the bias current (Ibias)
and will be split in half by Josephsons junctions. So now there is an upper 1⁄2 Ibias
and a lower
1⁄2 Ibias.
When applyling a small magnetic field (for example from neurons) a screening
current Iscreening will be observed. The Icurrent flows in the opposite direction of
the upper 1⁄2 Ibias and in the same direction as the lower 1⁄2 Ibias. In this way, the
lower half of the ring will now have a total current of 1⁄2 Ibias + Icurrent, whilst the
upper half of the ring will have a total current of 1⁄2 Ibias – Icurrent.
Eventually, the current in the lower half of the ring will be too big to maintain the
superconductivity; the critical current is exceeded. This causes the junction on this
side of the ring to momentarily become non-superconductive (resistive) and all the
current to flow through the upper side. This also causes the current in the upper half
to become too big (the critical current is reached) and for the junction to temporarily
become non-conductive (resistive). Now that there is a resistance, it follows from
the formula U=I*R that there also must be a voltage across the junctions. The
voltage is the signal that will be measured and used for imaging (see also figure 3).
This image shows what happens when a small
magnetic field is send through the superconductive
ring of a SQUID. It is visible that there will arise a
current and that it will be split in half by the ring.
Furthermore, it is visible that a screening current will
arise, that will run in the same direction as the lower
half bias current. Lastly, it is shown that a voltage will
arise in time.
Ultrasonic sound
8a) What is ultrasonic sound?
Ultrasonic sound is an oscillating sound pressure wave with a frequency over 20.000 Hz.
Humans are capable of hearing the frequency between 20-20.000 Hz. The frequency that
is used in diagnostic sonographic scanners is 2-18 MHz. A high frequency has a high
spatial resolution and less penetration. A low frequency has low spatial resolution and a
high penetration.
Advantages of ultrasonic sound:
- Free of radiaton risk
- Relatively inexpensive
- Real time images
- It is portable
8a) How is ultrasonic sound generated?
Ultrasonic sound makes use of the effect of
piezoelectricity: When piezoelectric crystals induce
electricity, pressure differences arises and pressure
differences induce electricity (see figure 1). This was
discovered by Jacques and Pierre Curie.
Piezoelectricity effect
An ultrasound wave is produced by a piezoelectric
transducer, the piezoelectric crystal. When there is an
electrical charge through the piezoelectric crystal, the
crystal expands. When the opposite electric charge
goes through the piezoelectric crystal, the crystal
contracts.
So to create an ultrasonic sound wave, short electrical
pulses are formed. These pulses make the filament
vibrate and create an ultrasonic pulse (see figure 2).
Generation of ultrasound
The velocity of the soundwave is determined by the material it goes through. There can
not be a vacuum, because the ultrasound needs molecules to move and creates kinetic
energy this way.
8a) How can a beam of ultrasonic sound be created and directed?
The creation and direction from ultrasonic sound
is made possible by phased array. The PA consists
of many transducers that can pulse separately.
These transducers are put next to each other and
the product of these transducers together is a
beam of ultrasonic sound. The beam can be
directed by the order of activating these
transducers (controlled by a computer). In figure
3 there is an example. If you activate the
transducers from the right to the left, the direction
of the beam will be to the left. So the angle of the
ultrasonic sound depends on the timing of the pulses.
Phased array
Beams of ultrasonic sound are used for medical ultrasonography. If you use multiple
probe elements, it is better steerable and it’s tightly focused. A disadvantage of PA is that
it is more complex and expensive compared to single-element ultrasonic inspection.
8b) How is the backscattered sound detected?
Ultrasound is a wave with a higher frequency than the upper limit of the human hearing
range. Ultrasound can be used for medical imaging. Tissue reflects the sound. Variation
in density of the different tissues leads to different reflections. A higher dense tissue
reflects more of the ultrasonic sound, whereas a lower dense tissue absorbs more and
thus reflects less.
Piezoelectric crystals detect the backscattered sound. Those crystals generate an electric
current when they are compressed (e.g. by a sound wave).
8b) How to generate a 2D image?
To create a 2D image the computer calculates the different amounts of reflection from
the ultrasonic sound. High reflectivity (sound wave that reflects on bone) is white in the
image, low reflectivity is grey (muscle and no reflection is black (water). By calculating
the difference in time between different incoming echoes you can measure the distance
of the tissue. A bigger difference means the tissue is farther away from the receiver. In
the 2D image the farthest tissue is displayed on the bottom and the closest on top.
8b) How can one reconstruct a 3D image from 2D images?
By comparing the contour of different 2D images the computer can make clear which
contours belong to the same structure. If the contours of two different images are very
close together, the surface between the two contours is proceeding towards the
“camera”. If they are farther apart the surface is more perpendicular. By using 3D
imaging techniques discussed in the first lecture (e.g. making some parts darker than
others) you can suggest 3D.
8b) Provide examples.
3D images are sometimes used for early detection of tumours. Most of all parents are
curious of what their baby might look like, and a 3D image makes it a lot easier to
imagine compared to a 2D image that also shows the internal organs.
What is Doppler ultrasound?
To be familiar with Doppler ultrasound we must understand the principles of the
Doppler effect. For example, the sirens of an ambulance sound different when it drives
toward you compared to when it moves away from your location. In ultrasound
something similar happens when you focus on the blood flow of arteries and veins.
Because the current either moves toward- or away from the sender/receiver, the echo’s
frequency changes. When the blood flows towards the receiver the frequency increases
and it decreases when it moves away from the probe. The frequency itself depends on
the speed of blood flow. By using software that measures the differences in frequencies
of the echoes we can determine the speed of blood flow, this is Doppler ultrasound. This
technique is used for example by cardiovascular surgeons if they wish to operate a
patient and they need to localize a clot.
Atomic force microscopy
8c) Describe the composition of an AFM.
1) Cantilever
2) Tip
3) Sample
4) Laser
5) Photodiodes
6) Piezoelectric tube
8c) What are the principles of AFM?
Atomic force microscopy makes use of a
cantilever with a sharp tip.
This tip is usually made out of silicon and its
radius is in the order of nanometers.
To scan the surface of certain materials, the
cantilever is brought close to this material.
This is done with a piezoelectric positioning
element. Being this close to the molecules of the sample, a lot of forces come in play.
Some forces attract the cantilever and some repulse the cantilever. Depending on the
material, these forces can be mechanical contact forces, Van der Waals forces, capillary
forces, chemical bonding, electrostatic forces and magnetic forces. The change in height
due to changes in force are measured by a laser which is pointed onto the cantilever
which then deflects in different ways due to these changes. The laser is deflected from
the cantilever into a detector which consists out of an array of photodiodes, this data is
being used to construct the final image of the sample.
There are multiple methods to scan a surface with AFM: the contact mode, the noncontact mode and the tapping mode. With the contact mode the tip scans the surface by
coming in close contact with the surface. This is also the common mode used in AFM.
While being dragged across the surface, the characteristics of the surface are measured
by deflections of the cantilever. The contact mode can also be used to test the thickness,
adhesiveness and strength of certain molecules on the surface, it can even be used to
test the strength of viruses. However this mode can damage the material by destroying
molecular bonds. The non-contact mode makes use of oscillation of the cantilever. Van
der Waals is the strongest force that influences the tip. This changes the amplitude of
oscillation of the cantilever, which can be measured, and the data can be used to create
an image. The tapping mode is used to scan the material without creating potential
destructive forces by dragging the tip over the surface (contact mode). Again, oscillation
is used with this method, but with a higher amplitude than the non-contact mode. The
closer the cantilever tip gets to the surface, the smaller the amplitude gets. Due to this
decrease in amplitude, the piezoelectric positioning element tries to maintain the
normal amplitude by moving the sample up or down via a feedback mechanism. These
changes in amplitude are measured by the deflection of the laser and can be translated
into an image.
8c) What are Van der Waals forces?
Van der Waals forces are forces that attract and repulse atoms, molecules and surfaces.
These forces are different from normal covalent bonds or normal ionic interactions, Van
der Waals forces work via electrostatic attraction and repulsion of atoms. They attract
when they are far apart, but they repulse each other when they are very close to each
other.
8d) Explain image formation with AFM in contact mode (constant force)
In this AFM mode the tip of the cantilever scans the sample while it is in close contact with
the surface of the sample. This is the common mode used in atomic force microscopy. A
force is set on the tip by pushing the cantilever against the sample surface (with a
piezoelectric positioning element). This repulsive force has a mean value of 10-9 N. The
deflection of the cantilever is sensed and compared in a feedback amplifier. If the measured
deflection is different from the desired value the feedback amplifier applies a voltage to the
piezoelectric positioning element to raise or lower the sample and restore the desired value
of deflection.
Sample surfaces are covered by a layer of adsorbed gasses consisting of (primarily) water
vapor and nitrogen. When the probe touches this layer, the cantilever is pulled by the
surface tension toward the sample surface. When the tip is too close to the surface
attractive forces can strongly pull the tip of the cantilever into the surface. To avoid this,
contact mode of AFM is done at a depth where the overall force is repulsive but the tip is
still in firm ‘contact’ with the solid surface below the adsorbed layers.
In contact mode AFM the tip of the cantilever is dragged across the surface of the sample.
Contours of the surface are measured by the direct deflection of the cantilever, or (more
commonly) by using the feedback signal which is required to keep the cantilever at a
constant position.
The probe moves in ‘x-direction’ (left to right) and scans the surface in the ‘y-direction’
(front to back). When a detail on the surface appears, the tip moves in ‘z-direction’ (up). This
is measured and a computer program will produce with these measurements a 3D image.
8d) Explain image formation with AFM in oscillation (tapping mode)
In this AFM mode the tip of the cantilever is not in contact with the surface of the sample,
but is oscillating up and down over the surface. The constant frequency of these oscillations
is determined by a piezoelectric element in the tip holder. This frequency is influenced by
different forces when the AFM tip is moving over the surface at a small distance, like: Van
der Waals forces, dipole-dipole interactions, electrostatic forces, etc. Through these forces
the amplitude or frequency of the tip is muted. The closer the tip approaches the sample,
the more the amplitude will decrease.
Just like in the contact mode, a detector registers differences in the amplitude. This detector
receives signals from (usually two) photodiodes, which collect the laser beam that is
reflected from a laser light on the surface of the cantilever. When the amplitude of the AFM
tip is changed by the surface of a sample, the reflection of the laser beam on the cantilever
will be changed which causes another distributions of laser light on the two photodiodes.
This change in distribution is detected by a detector and is proportional to changes in the
sample surface.
The advantage of the tapping mode over the contact mode is that the surface of a sample is
not damage by the tip. A limitation is that data obtained by the tapping is more difficult to
interpret because surface data is indirect measured by forces.
8d) What can be measured using these techniques?
The mechanical properties can be measured in the contact mode, by pushing with a certain
force on the surface of the cell. With this the breaking point of the cell membrane can be
measured. In either the contact mode or the tapping mode the cell surface can be imaged.
Also crystal structures can be measured of molecules such as gold.
A huge advantage of this imaging mechanism is, little preparation of the sample is
necessary. The measurements can be done in physiological conditions, whereas EM has to
be done in vacuum.
8d) Examples of AFM images
The image on the left shows the surface of the
bacterium S. Oneidensis. The image is in real life
approximately 5 µm. The little wires between
the red bacteria are nano-wires, produced by S.
Oneidensis. It is imaged using an AMF in noncontact mode.
On the right is an AMF image of the surface of
cerium oxide. As you can see using the scale, this
image displays the true atomic structure. It is
imaged using a conventional atomic force
microscope in non-contact mode.
Luciferase, Brainbow mouse
9a) Explain how luciferase tagging/labelling works
Luciferase is an enzyme, present in animals like the Aurelia aurita (moon jellyfish) and the
Photinus pyralis (North American firefly). This enzyme has different functions in these
animals. It is used to attract mates, to lure prey or for protection. Luciferase catalyzes the
Luciferin-reaction. The following image shows the reaction.
The reaction is catalyzed by firefly Luciferase (luciferase derived from the firefly) and takes
place in two steps. In the first step, Luciferin and ATP react to in luciferyl adenylate and PPi.
The luciferyl adenylate reacts with oxygen, into oxyluciferin in an electronically excited state
(carbon oxide, AMP and a hydrogen ion are produced). A photon is emitted as oxyluciferin
returns to the ground state. So there is light emission. The color of light that is emitted
depends on the type of Luciferin that is used. This chemical reaction is useful for observation
of biological processes. It is possible to determine the expression levels of a gene of interest
in different kind of cells during three steps. Firstly, luciferase needs to be expressed in the
novel host. This is done by transduction. Transduction is the process in which DNA is
transferred from one bacterium to another by a virus. Secondly, Luciferin is inserted into the
host cells/tissue by a dietary supplement. Finally, the experiment is normalized with the use
of a control gene. This final part needs to be done because the transduction of Luciferin is less
than 100%.
9a) Can Luciferase and Fluorescent protein tagging/labelling be used only
qualitatively, or also quantitatively?
Luciferase and Fluorescent protein tagging can be used qualitatively and quantitatively. With
quantitative use we can observe a few characteristics:
- Gene expression levels
- Protein concentration
- ATP assays
When Luciferase is overexpressed and it becomes abundant in the cell, the ATP becomes
the limiting factor. In qualitative use of luciferase and fluorescent protein tagging, the
presence or absence of a protein can be observed.
The difference between Luciferase and Fluorescent is that Fluorescence absorbs light
and when needed it emits the same light again. Luciferase produces it’s own photons out
of Luciferin, ATP and oxygen. Because of this, controlling the input controls the output of
a Fluorescent protein. When you compare the two, the Luciferase tagging/labelling is
better for quantitative observations. For qualitative observations the Fluorescent
protein tagging/labelling is favourable.
9a) Give some luciferase examples and practical use
If Luciferase is transcribed together with a protein of interest, gene expression and protein
concentration could be measured. Another quantitative use of Luciferase is ATP assay in
vitro. Luciferase can act as an ATP sensor protein. For the emission of light, Luciferase needs
ATP. As long as there is ATP, there will be light emitted.
A qualitative use of Luciferase, the presence or absence of a protein can be detected.
Luciferase could therefore be used to image cells or subcellular structures. Because
Luciferase yield a low signal, it is more appropriate to use Fluorescence as a method for
qualitative measurements.
9b) What is a brainbow mouse? How is it created?
De Brainbow mouse is een benaming voor een muis waarvan de individuele neuronen
gekleurd zijn, zodat neuronen van elkaar onderscheiden kunnen worden. De kleuring
wordt veroorzaakt door het gebruik van verschillende soorten fluorescente eiwitten
(XFP’s). In de brainbow mouse worden er meestal vier verschillende soorten XFP’s
gebruikt. Namelijk het Green Fluorescent Protein (GFP), Yellow Fluorescent Protein
(YFP), Red Fluorescent Protein (RFP) en Cyan Fluorescent Protein (M-CFP). Genen
of eiwitten kunnen met deze fluorescente eiwitten gelabeld worden, waarna de
expressie in vivo kan worden aangetoond.
De kleuring van de neuronen is gebaseerd op recombineren met behulp van het
Cre/LoxP recombinatie systeem. Cre is een gen uit een bacteriofaag dat codeert voor
het recombinase enzym. Recombinase zorgt ervoor dat er een inversie of een insertie
plaatst vindt op de ‘Lox plaatsten' in het DNA, die coderen voor verschillende kleuren
XFP's. Om het Cre/Lox systeem te kunnen gebruiken, moet er eerst een construct met
het Cre/lox systeem worden ingebracht in een cel (figuur 1). Recombinase zal
vervolgens willekeurig inserties en deleties uitvoeren op de Lox plaatsten. Het
resultaat is een willekeurige expressie XFP's die door licht kunnen worden
aangeslagen. Bij het absorberen van de juiste golflengte licht, raakt het eiwit in een
aangeslagen toestand en na de relaxatie zendt het eiwit langere golflengtes uit, die
zichtbaar zijn als oplichtende structuren. De hersenencellen van een brainbow muis
bevatten meerdere constructen, waardoor cellen verschillende kleuren tegelijk
uitzenden. In totaal kunnen er ongeveer 100 verschillende soorten kleuren ontstaan.
There also is another brainbow constructs which contains an inversed RFP and noninversed CFP, which can flip under the control of the CRE/loxP system, creating
either one of the two coulors (red or blue). Another construct is the multiple insertion
of the brainbow construct, which will create not 2 or 3 colors, but ~100 colour
combinations. All of these constructs are only expressed in the brain because they are
under the control of the neuron specific Thy1 promotor.
Figuur 1. Voorbeeld van een Cre/Lox construct. De promotor is de promotor van een gen waarin men
geïnteresseerd is. De kleuren stellen verschillende XFP's voor. Op de Lox plaatsen kunnen inserties of
deleties plaatsvinden. In dit voorbeeld wordt er een GFP afgeschreven en zal de cel een groene kleur
krijgen.
Voor het bestuderen van neuronen van een brainbow (mouse) kan een confocaal
microscoop gebruikt worden. Deze microscoop heeft de voorkeur, omdat het anders
lastig is om verschillende kleuren XFP's in één plaatje weer te geven. Een normale
fluorescentie microscoop kan maar één soort XFP aanslaan, om een tweede XFP aan
te slaan, is er aan andere laser nodig. De confocaal miscoscoop heeft verschillende
lasers en kan daardoor makkelijker de verschillende XFP's laten zien (Figuur 2).
Figuur 2. Verschillende neuronen, gelabeld en gekleurd door middel van XFP’s.
9b) Provide examples of its use.
De Brainbow kleuringsmethode biedt mogelijkheden om de functie en interactie
tussen neuronen nader te bestuderen. Zo kunnen interacties tussen individuele
neuronen bestudeerd worden, maar kunnen, aan de hand van een neurale map, ook
neurologische en psychische stoornissen worden geanalyseerd. Het bestuderen van
neuronale connecties en mogelijk deficiënties hierin kunnen bijdragen aan een beter
begrip van verschillende hersenziekten.
Confocal scanning microscopy
10a) How is a confocal microscope composed? What is the purpose of the
LASERs?
Confocal scanning microscopy is an imaging technique that makes use of a laser to excite
fluorophores in the specimen you want to see. Fluorophores are chemical compounds
that can re-emit light after being “excited” with a light beam, in this case a laser. The
emitted light always has a higher wavelength (so a lower energy) then the excitation
light.
10a) What are the diaphragms (“pinholes”) for?
This technique of confocal scanning makes use of pinholes
to create the image. Only a small part of the specimen is
being collected by the camera each time. The first pinhole
creates a very thin beam of light that scans the specimen.
The second pinhole makes sure that only the light that is in
focus will be measured by the camera. This is to make sure
no background haze will be seen in the resulting image, so it
will be clearer and better.
10a) What is a dichroic mirror and what is its function?
What about the excitation filter and the fluorescence
barrier filter?
Excitation filter and dichroic mirror
The laser passes through the excitation filter and on a
mirror, called a dichroic mirror. The excitation filter makes sure that only selected
excitation wavelength is let trough when using multiple lasers. Via the dichroic mirror
the laser beam is send onto the wanted place on the specimen, but the more important
function of this dichroic mirror is that it can reflect light that is shorter than a certain
wavelength, but lets light with a higher wavelength pass through. Because the emitted
light of the fluorophores in the specimen always has a higher wavelength, the mirror is
capable to let only the emitted light pass through.
Fluorescence barrier filter
The fluorescence barrier filter (emission filter in the figure) can filter out the
remaining excitation light from fluorescent light. Hereby, unwanted excitation light
such as ultraviolet light is blocked.
The emission filter (aka barrier filter or emitter) attenuates all of the light transmitted
by the excitation filter and very efficiently transmits any fluorescence emitted by the
specimen. This light is always of longer wavelength (more to the red) than the
excitation color.
Confocal microscopes are used to increase optical resolution and contrast of micrograph
by using point illumination and a spatial pinhole to eliminate out of focus light in
specimens that are thicker than the focal plane.
Examination of thick specimens, intracellulair studies, and in studies where the threedimensional structure of the sample is important.
10a) Explain the principles of fluorescence.
Fluorescence is the emission of light by a substance that has absorbed light or other
electromagnetic radiation. It also occurs when molecules are excited to higher electronic
states by energetic electron bombardment. It is a form of luminescence. The emitted
light has a longer wavelength, and therefore lower energy, than the absorbed radiation.
10a) What is photobleaching?
Photobleaching is the photochemical destruction of a fluorophore. Photobleaching may
complicate the observation of fluorescent molecules, since they will eventually be
destroyed by the light exposure necessary to stimulate them into fluorescence.
10b) Describe the principles of object scanning
Traditional fluorescence microscopy tends to enlighten the whole specimen whereas
confocal microscopy uses a pinhole as well as point illumination to eliminate any light
not coming from within the focal plane from reaching the photodetector/camera,
resulting in high resolution and contrast. With this technique thicker specimens can
be used.
The normal order of components of the microscope is: laser, pinhole, excitation filter,
dichromatic mirror, objective, dichromatic mirror, filter, pinhole, detector.
Because only one point can be sufficiently scanned due to the properties of point
illumination and the pinhole, confocal microscopy is not the fastest of scanning
techniques. Scanning multiple sections however allows a form of 3D imaging.
10b) Which parts of the
microscope are moved? How?
With laser scanning microscopy
an image is created by a focused
laser beam moving across the
sample in a raster pattern. The
laser beam is moved across the
sample by two moving mirrors.
The mirrors are controlled by
galvanometer motors. The sample
stays in the same place during the
creation of the entire image (see
figure 1).
Figure 1. structure of the microscope
10b) The movement has consequences for some parts of the beams but not
for all. Which parts?
The exciting LASER beam and thus, the returning LASER beam are redirected by
the rotating mirrors. What this means is, that the beams are moved across the
specimen, but, as soon as they return via the second mirror, the beams follow the
same path as they always do. The exciting beam, reflected by the dichromatic mirror
will stay in the same place, as will the returning beam through the dichromatic mirror
on the detector. The LASER and the PMT never move.
10b) What is de-scanning?
The laser beam is reflected by the dichroic mirror. The beam is then pointed at the
specimen and moved across the specimen by the rotating mirrors. When the laser
beam has hit the specimen, the wavelength changes. The light that travels back from
the specimen will now pass the dichromatic mirror because of it larger wavelength. It
will then reach the photomultiplier detector. This process is called de-scanning.
10b) What is the purpose of the photomultiplier?
During scanning, the photomultiplier
measures the intensity of the
fluorescence in combination with the
current position of the mirrors. So
there will be specific information with
specific coordinates. With this
information an image can be created.
The photomultiplier also enhances the
photons obtained from the sample,
because only a little light gets past the
photocathode. (shown in figure 2)
Figure 2. Build-up of the photomultiplier tube
10b) Why would one sometimes use more than one LASER?
If you want to detect more than one protein within a sample you can you use multiple
LASERS. The proteins need to be colored with different kinds of fluorescent proteins
so they can be separated from one another. The used LASERS need to have
different wavelengths to excite different colors of light. The use of multiple different
blocking filters is needed to filter certain wavelengths of light onto the different
photomultiplier detectors. Multiple detectors are needed because the detector makes
no distinction between the different wavelengths of light.
Figure 3. Result of usage of more than one LASER.
In situ hybridisation, Immunocytochemistry, Radioactive
labeling, Fluorescence
11a) What is in situ hybridisation, what is its use, provide examples.
11a) What is immunocytochemistry, what is radioactive labeling, what is its
use, provide examples.