Lecture 1: Basic principles pt.

Light microscopy  includes fluorescence microscopy (2-photon, confocal and super resolution)

1. What is the lowest possible refractive index?

Ni=c/vi --> 1 bc speed of light in medium cannot be larger than c

2. What is diffraction?
- Bending of a wave as it passes through a hole or around an obstacle.
- If light consists of parallel rays, they would travel through a small pinhole and make a small,
bright spot on a nearby screen --> spot larger than the pinhole and varying in brightness
- The pinhole somehow affects the light that passes through it.
o Diffraction is proportional to the ratio of wavelength to width of gap.
o The longer the wavelength and/or the smaller the gap, the greater the angle through
which the wave is diffracted.

3. What is light interference?

- Combination and interaction of 2 different waves resulting in changes of amplitude
o Constructive
o Destructive

4. What is magnification?
- Magnification = ratio of the size of the image to that of the object. This means, that an object
of any size is magnified to form an enlarged image
- Magnification is defined by the magnification by eyepiece (10x) x the magnification by the
objective

5. Define spatial resolution. How does this term apply to light microscopy?
- Light from a point source is diffracted by the objective to form an Airy Disc/Point Spread
Function --> Resolution describes the minimal distance of two points that can be
distinguished.
- Numerical aperture (NA) of the objective determines its resolution = how much light enters
the microscope and resolve fine specimen details:
o NA=(a)sin(µ) --> µ = theoretical maximum 90º – reality is 72º

6. How do we make the details visible?

 - Contrast --> label samples show internal cell organization
- Phase shift using (properties to do that= intensity, fq and phase --> humans don’t perceive
phase shifts. We can only see changes in amplitude or wavelengths
o Microscopes: changes phase shifts (caused by light and specimen interactions) into
amplitude shifts

7. Brightfield: light source, objective, condenser

- Principle: Light is transmitted through the sample and absorbed by it
- Application: Very little contrast in unstained specimens. Only useful for specimens that can be
contrasted via dyes (technique, e.g. Gram stain, methylene blue, fuchsin , etc.). With a bright
background, the human eye requires local intensity fluctuations of at least 10 to 20% to be
able to recognize objects.
- Disadvantage: most cells and tissue are transparent. They need to be fixed and stained to
show contrast which usually means that this is not compatible with live cell imaging

8. Darkfield --> light source, objective, condenser and light stop (annular stop)
- Principle: The illuminating rays of light are “blocked” and directed through the sample from
the side. This is achieved by putting a dark disk (light stop) into the condenser. This hinders
the main light beam to enter the objective. Only light that is scattered by structures in the
sample enters the objective.
- Most structures inside the specimen do not scatter much light and will appear dark. Only
highly refractive organelles that scatter large amounts of light will appear bright.
- Application: marine organisms such as algae, plantkon but also blood cells

9. Phase contrast --> light source, objective, condenser light stop (annular stop)
- Principle: Incident light is out of phase with transmitted light  interference lens=optical
tricks to translate phase shifts into grey values (intensity/amplitude changes).
- Application: unstained specimens (cultured cells)
- A phase plate (ring) is mounted in order to selectively alter the phase (and amplitude) of the
surround wave. We also want to change the amplitude because it is usually too bright and not
enough contrast can be generated
- Positive phase contrasts rely on destructive interference  systems selectively advance the
phase of the surround wave relative to that of the diffracted wave.
- Negative phase contrast relies on constructive interference

10. Write in a couple of sentences what is the numerical aperture of an object and why is it
important in microscopy
 - Light from a point source is diffracted by the objective to form an Airy Disc/Point Spread
Function/diffraction pattern --> Resolution describes the minimal distance of two points that
can be distinguished.
- Airy disk is the region enclosed by the first minimum of the airy pattern and contains
approximately 84 percent of the luminous energy,
- Numerical aperture (NA) of the objective determines its resolution = how much light enters
the microscope and resolve fine specimen details:
o NA=(a)sin(µ) --> µ = theoretical maximum 90º – reality is 72º

11. Polarization contrast  it requires a polarizer and an analyzer

- Principle: Only when the vibration direction of the polarized light is altered by a sample
placed into the light path, light can pass through the analyzer. The sample appears light
against a black background.
- Application: Polarization contrast is used to look at materials with birefringent properties.

12. Differential Interference Contrast (DIC) --> polarizer and an analyzer two birefringent prisms
- Principle: requires polarized light for illumination and additional light shearing (Nomarski )
prisms to exaggerate minute differences in specimen thickness gradients and refractive index.
- Pseudo 3D images  lipid bilayers (difference bw aqueous and lipid phases of the cell)
Lecture 2: Basic principles pt. II
13. Explain the Rayleigh Criterion
When the distance between the Airy disks is increasingly reduced, a limit point is reached (Rayleigh
criterion) when the principal maximum of the second Airy disk coincides with the first minimum of
the first Airy disk.

14. Write a couple sentences about resolution and how is determined? How can resolution be
improved?
- If the two image points are far away from each other, they are easy to recognize as separate
objects and it is determined by the Rayleigh criterion.
- Problem is that the image of a point source is always larger than a point source itself
o Increase resolution by lowering the wavelength or higher the NA  immersion liquid
(oil, water, glycerol or silicone oil)
 15. Is the spatial resolution in the Z (axial) direction the same as that of the XY (lateral)? (one correct
answer)
a. Resolution is the same
b. Z-axis resolution is better than the XY-axis resolution
c. Z-axis resolution is about 3x worse than the XY-axis resolution  since lateral
resolution is inversely proportional to the numerical aperture of the system NA, while the
axial resolution is inversely proportional to the squared numerical aperture NA2
d. Depends on the sample and imaging depth

16. Order from shortest to longest wavelength: 1) Photon, 2) Electron, 3) X-ray  2,3,1

17. How can bit depth affect image file size? Why is it mostly sufficient to store the image with 8-bit
depth? Explain in 2-3 sentences.
The bit depth of an image determines which pixel values it can contain. A higher bit depth leads to
larger file sizes, and potentially slower processing. The amount of light detected per pixel might be so
low that thousands of possible values are not required for its accurate storage, and 8 bits (or even
fewer) would be enough.
 18. Which of the following formats is appropriate for storage of imaging data? (More than 1, less
than 4 answers possible)
a. .gif
b. .TIFF, grey lookup table
c. .JPEG
d. .TIFF, color lookup table
LUTs (Lookup table, color maps): Changing the LUT does not change pixel values (it only changes how
an image will appear)

19. IMAGE FILE FORMATS

Lecture 3: Fluorescence microscopy

20. Explain the basis of fluorescence (draw a scheme if necessary)
- Fluorophors (= Flurochromes )): Molecule or group of a molecules
that absorbs energy if a photon arrives that matches a possible
electronic transition within the molecule:
1. light energy is absorbed, and electron is promoted to a higher
energy orbital
2. “something” happens inside the molecule
 3. the energy is re emitted in form of a photon of a different (=longer) wavelength

21. Explain FRET and give one example

When 2 molecules are in a close distant (1-10nm) and coupled with fluorescent proteins. When
there is absorption of energy from the one molecule (donor), the energy transfers from the donor
electrons to the acceptor electrons, which in result in re-emiting the energy in longer wavelength
(shorter energy).
There is loss of fluorescence due to other electron energy transfer pathways

22. FRET works best at which distance? (One correct answer)

a. 5 nm
23. Name three methods (each) for delivering synthetic and genetically encoded fluorophores

24. Main inconveniences/problems fluorescent microscopy

25. Name similarities and differences between Photomultiplier Tube (PMT) and Hybrid Detector
(HyD)  both convert photons into electric signals and amplify the signal

- PMT has a large dynamic range, lower signal to noise ratio, amplification via 10 dynodes
- HyD is faster, better signal to noise ratio for weak signals, BUT can saturate easily. Avalanche
photodiode
26. Mention three advantages and three disadvantages of confocal fluorescence microscopy

27. Why is penetration depth higher in 2-Photon microscopy? Explain in one short sentence.
- Two-photon microscopy can penetrate deeper into tissue (500 μm−1 mm) than confocal
microscopy because the excitation wavelength is about twice as long as the usual
wavelengths used. Light with longer wavelengths scatters less, allowing deeper penetration
into tissue.

28. Mention three advantages and three disadvantages of 2-Photon fluorescence microscopy
- Another advantage is that with a lower energy  deeper and nontoxic image acquisition
 29. Imagine you are trying to image your fixed sample at the confocal microscope with appropriate
pinhole size and the signal is dim. What would you change first in order to increase the signal?
Please describe which parameter you would change and why.
- Increasing the gain makes the PMT more sensitive and so your sample looks brighter.
Reducing the offset reduces the background level.

Lecture 4: Optical sectioning

30. What distinguishes epifluorescence illumination from transmitted (diascopic) illumination?

- In epifluorescence (episcopic illumination), the objective lens is used as both the illumination
condenser light from the light source goes through the objective to the sample and the
fluorescent light collector emitted light from the sample goes through the objective to the
detector
*In both upright and inverted microscopes, you can use both transmitted and epifluorescent
illumination

31. Main inconvenience of simple epifluorescent microscopy. How can you overcome this?
Problem is out of focus light!
- Image degradation due to light scattering (blurring effect, noise, etc)
- Not such a problem with cells in culture, but is obvious in thick samples
To overcome this, we use optical sectioning (division of 3D specimen into several 2D focal planes) 
you can use microtome to section the sample but in vivo there are other techniques  STANDARD
FLUORESCENCET MICROSCOPE DOES NOT PROVIDE OPTICAL SECTIONING

32. Optical sectioning methods

33. Why are laser the best option for confocal microscopy?
In confocal we need higher energy bc of the pinhole  laser (focusability + energy). Smaller pinhole =
higher resolution
 - Disadvantage  multiple laser lines to cover multiple fluorescence spectra

34. Advantages of confocal

- Rejects out-of-focus light
- Collects series of images from different focal planes
- Can assemble images to give 3D representation

35. How to image a specimen if the light source is focused to a single spot? Disadvantages?

36. What is to be adjusted regarding the laser in confocal imaging?

- Appropriate laser line
- Power adjustment
- Scanning speed (pixel dwell time)  Very small values (µs range) defined by scan frequency.
It is important for signal intensity => higher pixel dwell time = higher signal but slower image
acquisition and more bleaching
- Averaging  helps reducing the noise. Try a few values and see what the minimum that gives
you a good image Line averaging somewhere between 2 and 8 are the most common choices.

37. What is to be adjusted regarding the pixel size in confocal imaging?

- It is defined by zoom and pixel number (quick scanner 512x512// high resultion 1024x1024)
o Change zoom  image are changes but nº of pixel stays the same (higher energy
deposition  Bleaching)
o Change pixel number  image area stays the same but nº pixel changes (slower
scanning)
 38. What is the Nysquit-Shannon sampling theorem and how can this help to state the pixel size to
avoid under/over sampling?
- The Nyquist sampling theorem states that the sampling frequency must be at least twice or
greater than twice the bandwidth of the input signal to reconstruct the original input from the
sampled data  pixel size is fq and input signal and input signal is resolution
o 2 pixels per resolution at least (2.3 is better)  needs to be match for each objective
o Same calculation regarding optical thickness when taking x-stacks

39. Nyquist theorem: You want to get a z-stack in confocal microscopy with a resolution of 300 nm
a. How do you sample in the XY-axis to avoid over/undersampling? (one correct answer)
i. 150 nm
ii. 200 nm
iii. 450 nm
iv. 900 nm
b. Which thickness do you choose in the Z-axis?
i. 150 nm
ii. 200 nm
iii. 450 nm
iv. 900 nm

40. What is to be adjusted regarding the PMT in confocal imaging?

Gain and offset. They affect the sensitivity and background level of the detectors (the
PMTs
- Increasing the gain makes the PMT more sensitive and so your sample looks brighter.
Reducing the offset reduces the background level.
 - Microscopes usually have a special display mode (lookup table) that helps you set these.
Adjust the gain so just a few pixels are the max colour, reduce the offset so the background is
about 50% the 0 colour. This ensures you have the full range of brightness within your image.

41. What is to be adjusted regarding the pinhole in confocal imaging?

Pinhole size:
- It affects the brightness, optical slice thickness and resolution

42. What is spinning disk confocal imaging?

- The spinning disk confocal microscope scans the specimen with thousands of spots of light
simultaneously so images can be captured more rapidly than on a spot scanning system 
through a two-disc system
- The microlenses focus the excitation light with greater efficiency onto the imaging pinholes
- The spinning disk system is particularly well suited for studies that require capturing images of
fluorescent specimens rapidly or for long periods of time

43. Light sheet microscopy

 44. Deconvolution
Computationally intensive image processing technique (alternative for confocal smtimes but you can
combine both things  usually is for standard widefield fluorescence microscope).
- It is a techique to get rid of out-of-focus information
o Noise. Quasi-random disarrangement of detail  If we can predict noise distribution,
that means that we can also get rid of it. Deconvolution and filtering does that.
o Scatter is a random disturbance of light induced by passage through regions of
heterogeneous refractive index within a specimen  difficult to get rid of it
o Glare is a random disturbance of light but occurs in the optical train  difficult to get
rid of it
o Blur is described by a nonrandom spreading of light that occurs by passage through the
imaging system optical train caused mainly by diffraction  BASIS OF
DECONVOLUTION IS TO PREDICT THE DIFFRACTION INDUCED BLUR

Get the PSF  Providing information like excitation and emission wavelength peaks and the NA of
the used objective, this theoretical value is estimated by a computer algorithm.

SUMMARY OF MICROSCOPY
Lecture 5: Super-resolution techniques and CLARITY
45. What kinds of super-resolution techniques exist?
- Structured illumination microscopy (SIM). Generation of an interference pattern bw spatial
fq in the probe and a grating that shifts spatial fqs down to lower fqs through the objective
(indirect decrease of PSF).

- Reversible saturable optical fluorescence transition (RESOLFT). Sharpening or decreasing size

of PSF by reducing its airy disc diameter  STED illumination

*STED stimulated emission depletion microscopy  Laser scanning technique based on

increasing resolution through de-excitation of excited fluorophores via stimulated emission
 using a red shifted light beam with a local intensity 0 in the center (donut mode beam)  turn
off the fluorophores in the outer part of the focus

Single –molecule localization microscopy (SMLM).

- Make use of the fact that single fluorophores can be

located with high precision if sparse enough (and S/N
ratio excellent, etc.)  IMPROVE PSF OPTICALLY, PALM
and STORM

- STED’s problem is photobleaching, PALM’s problem is

the long acquisition time, so why not combining both
methods…. called MINFLUX (named after the few
photons than can be used…)
 46. CLARITY: ADVANTAGES AND DISADVANTAGES

NOT LIFE TISSUE

47. Which of the following statements about CLARITY are correct

a. CLARITY can only be used for anatomical but not for physiological experiments
b. CLARITY is a technique to image long distance projections in the brain
c. CLARITY has the same high resolution as electron microscopy
d. CLARITY requires objectives with a long working distance of several micrometers

48. Which super-resolution technique reduces (optically) the size of the Point Spread Function to
obtain a better resolution? (one correct answer)
a. CLARITY
b. STED
c. SIM
d. PALM
e. dSTOM

49. How can super-resolution increase the spatial resolution in XY-axis compared with conventional
light microscopy?

By affecting the PSF  xy resolution form 200nm to 20-100 nm

Lecture 6: Advanced fluorescence microscopy

50. Idea + pros and cons of sensing Glu release through iGluSnFR
 - Idea: instead of measuring pre-or postsynaptic calcium as an activity signal measure the
transmitter directly  biosensor expression in synaptic cleft (not FRET based)  fusion with
GFP
o Measure presynaptic output and postsynaptic input and correlate with overall activity
o Measure synaptic transmission on subcellular compartments

51. Why iGABASnFR?

- Cl- snesors have poor biophysical performance + Cl- concentration doesn’t change too much

52. Sensing membrane voltage: ACE

53. Describe briefly the mechanism of fluorescence in a fluorochrome

Fluorochromes will only fluoresce if they are illuminated with light of the corresponding wavelength.
The wavelength depends on the absorption spectrum of the fluorophore and it has to be ensured
that an appropriate quantity of energy is delivered to elevate the electrons to the excited state.
After the electrons are excited they can dwell in this high energy state for a very short time only.
When the electrons relax to their ground state or another state with a lower energy level, energy is
released as a photon. As some of the energy is lost during this process, light with an increased
wavelength and lower energy is emitted by the fluorochrome compared to the absorbed light.

54. What happens to fluorophores after they are excited with visible light (in single photon
absorption)? (one selection)
 a. The energy of the fluorophore is initially increased and then as the energy state
increases, the light is emitted at shorter wavelengths
b. The longer that they are excited with light the more different wavelengths they emit
c. The longer they are excited with light, the longer wavelength of the light they emit
d. The energy of the fluorophore is initially increased and as the energy state relaxes, light
is emitted at longer wavelengths

55. What is the purpose of halorhodopsin expression in neurons in experimental neuroscience? (one
correct answer)
a. Inhibition of cells because it is a light-driven chloride pump
b. Excitation of cell because it is a non-selective cation channel
56. Issues regarding optogenetic tools
- Delivery of these genetically encoded “tools” into the target neuron(s) (similar strategies as
for FPs)
- Sufficient excitation light to optimally control neurons via the light-induced channels (i.e. deep
inside the brain tissue)
- Problematic for restoring vision

57. Optopharmacology and photopharmacology

- Uncage neurotransmitters by excitation to activate

neurons (Glu-cage fusion)

- To make it reversible and cell type specific

photopharmacology
o Tethered (t-)toxins
Lecture 7: Cell-labelling techniques
58. What is expansion microscopy
Physically magnify a preserved biological specimen by synthesizing a dense, cross-linked network of
swellable polyelectrolyte hydrogel throughout such a specimen This in turn can smoothly and
isotropically expand biomolecules or labels away from each other after chemical processing. After
such physical magnification, molecules in a diffraction-limited region are separated in space to greater
distances, and therefore can be resolved even by conventional diffraction-limited microscopes.

59. Iterative expansion microscopy

Here we describe iterative expansion microscopy (iExM), in which a sample is expanded ∼20×. After
preliminary expansion a second swellable polymer mesh is formed in the space newly opened up by
the first expansion, and the sample is expanded again. iExM expands biological specimens ∼4.5 × 4.5,
or ∼20×, and enables ∼25-nm-resolution imaging of cells and tissues on conventional microscopes.
We used iExM to visualize synaptic proteins, as well as the detailed architecture of dendritic spines, in
mouse brain circuitry.

60. Which of the following are correct? (multiple answers possible)

a. Conventional dyes are sued in PALM
b. Fluorescent proteins are used in PALM
c. Conventional dyes are used in STORM
d. Fluorescent proteins are used in STORM

- In STORM (Stochastic Optical Reconstruction Microscopy), fluorescent organic dyes (cyanines,

rhodamines, …etc.) together with a specific imaging buffer (abbelight’s buffer) are used to
allow blinking of the fluorescent molecules.
- In PALM (Photo-activated Localization Microscopy), photoactivatable, photoconvertible or
photoswitchable proteins are used (ex: PA-GFP, PA-mCherry, mEOS, mMAPLE, …etc.).
- In PAINT (Point Accumulation for Imaging in Nanoscale Topography), reversibly-binding
fluorescent probes are used (ex: Nile Red).

61. Describe in a few sentences the differences between PALM, dSTORM and STORM.
All 3 are SRM techniques sharing the same basic principle.
The main difference between PALM and STORM/dSTORM is the fluorophores used for the
experiment and the mechanism of switching between the bright and dark states. PALM uses photo
switchable/convertible fluorescent proteins (FPs), whereas STORM/dSTORM uses organic dyes as
fluorescent
probes for imaging.
- PALM (Photoactivated Localization Microscopy): photoactivatable, photoswitchable and
photoconvertible proteins that change their structure when exposed to UV light
- STORM (Stochastic Optical Reconstruction Microscopy): uses a pair of activator reporter dyes,
short wave dye for activation and longer wavelength dye for detection
- dSTORM: dyes without activator, so the reporter dye is activated without the help of activator
dye, requiring for an imaging buffer with reducing agents to recover the excited dye back to
ground state.
What is the difference between STORM and dSTORM?
dSTORM microscopy is the "direct" variant of STORM that makes use of fluorophores that are very
bright, have a high rate of photoswitching and exhibit minimal photobleaching.
 62. Imagine you are a PhD student in a Tübingen neuroscience lab and plan an experiment involving
imaging of voltage-gated potassium channels in living neurons. Which protein labeling method
would you choose and why? Please name at least two advantages and at least two
disadvantages of the chose method.
ACE?¿

63. Imagine you are supposed to start an experiment which involves comparing STED and dSTORM
imaging of neurofilaments in cultured primary mouse neurons. After cell fixation, you will do
immunocytochemistry staining with an anti-neurofilament primary antibody and a fluorescently
labelled secondary antibody. Since your lab freezer has a large collection of secondary antibodies
i.e. antibodies conjugated with different fluorophores (dyes), what is important to keep in mind
when choosing the most optimal dye(s) for such an experiment? Please describe it in a couple of
sentences. CHOOSE DYES

- When choosing dyes for STED, one must be careful to select exceptionally stable fluorophores
whose properties match the depletion laser lines available ((592 660 775 nm)
- For STORM you need a pair of orange and red emitting carbocyanine dyes, Cy 3 and Cy 5
combined to form what is referred to as an activator reporter pair or more colloquially as a
“dye pair”. Cy5 fluorescence regulated reversibly by Cy3  activation at 647nm
- dSTORM  dyes blink without activator. In many cases, activation of the reporter may be
accomplished with a high energy laser and without the aid of a proximal activator dye.
o For this to happen u need special imaging buffers.
64. DNA PAINT
Transient binding of dye labeled DNA strands (imagers) to their
complementary target sequence (docking site) attached to a molecule
of interest. The transient binding of imager strands is detected as
'blinking', illustrated by the intensity versus time trace.

65. Probes for PALM

 66. How to label proteins for SRM?
- Fusion proteins (fluorescent proteins)
- Organic dyes (IF, Enzyme mediated protein labeling…)
o IF. Cons  Ab are big and usually not compatible with life imaging
 Alternative  nanobodies (only heavy chain). You can use them for SRM and
live imaging
o Protein/peptide directed labeling (SPONTANEOUS). Halotag or SNAP/CLIP-Tag
o Enzyme mediated (not spontaneous – enzyme mediated = substrate and takes time
bro)
o Minimally invasive tags. Aminoacid based labeling (Unnatural Aminoacids – UAAs) 
also the fluorophore is closer to the target
*UAAs:
- Expanded genetic code relies on additional components (such as UAA specific tRNA, UAA specific
tRNA synthetase and a specific codon)
---> Unique codon=UAG (stop codon)

OTHER TECHNIQUES (APTAMERS and QUANTUM DOTS)

Lecture 8 and 9: Electron microscopy

67. Why electron microscopy?  ultrastructure (important in diagnostic too)

To obtain higher resolution (Abbe-Limit)  decrease wavelength
Accelerate particles to analyze materials instead of photons
68. Name two differences between TEM and SEM
TEM
- Contrast in EM: higher the bigger the nuclei of the atoms are
- Magnification in TEM achieved by changing the strength of the magnetic field in the magnetic
lens and thus changing the focal length (IN SEM electromagnetic lenses are only for focusing)
- Image acquisition through fluorescent screen covered with zinc sulfide  emits yg light upon
exposure to the electron beam
- Creating digital data  CCD camera

69. SAMPLE PREPARATION IN EM

1) Fixation. GA + PFA (important to control pH, Tª, buffer, size of the tissue or osmolarity)
 a. Glutaraldehyde  crosslinking proteins
b. PFA  more penetration
2) Dehydration  contrast enhancement (more contrast adding uranyl acetate and lead citrate)
3) Embedding in resin polymerization with warm or cold temperature or UV
4) Ultrathin sectioning  ultramicrotome
5) Electron microscopic analysis

70. Freeze fracture  USED ANALYZE THE MOLECULAR ANATOMY OF BIOLOGICAL MEMBRANES

71. High pressure freezing Why?

The theoretical basis of HPF is the principle of Le Chatelieraccording to which the volume of water
increases when it freezes. High pressure inhibits the expansion of water during freezing and thereby
inhibits crystallisationby altering the following freezing properties of water: (1) lowering of the
freezing point, (2) reduction in the rate of ice crystal formation, and (3) slowing of the growth of ice
crystals.
- Replace chemical fixation with physical fixation
- Overcome technical artifact  unspecific lamella splitting in myelin (myelin ultrastructure is
preserved)
 72. Name two techniques for labeling proteins in electron microscopy and a problem for each
- Immunogold labeling (primary and secondary ab)  challenge morphology of the tissue as
good as possible and at the same time maintain the ability of antigens to react with
antibodies
o Pre-embedding  Ag preservation but loss of ultrastructure
o Post-embedding  less recognition of Ag
* Cryo-fixation with subsequent post-embedding e.g., deep temperature embedding or high-pressure
freezing) may solve some of the problems with antigen-recognition and structural preservation
- Horseradish peroxidase for pre-embedding labeling in E

73. Where are the membranes torn apart in freeze fracturing?

a. In between two adjacent double layers
b. In the hydrophobic core
c. ??
d. ??

74. Which of the following influences the image quality significantly in TEM?
a. The choice of embedding resin
b. The crosslinking
c. Staining of membranes with heavy metals
d. ??

75. Sample preparation for SEM

- Non conductive elements need gold coating

76. Backscattered and secondary electrons

77. Imaging in SEM
Unsorted
78. Imagine you are already a PI of a Tübingen neuroscience lab and plan an experiment to analyze
the connections between neighboring neurons in a local circuit (distance of neurons ~100 μm) in
the brain. For your scientific questions both live and dead (fixed) brain tissue would work. Name
one imaging method with two advantages and two disadvantages.

- Light microscopy  so we can actually the connections between neurons

Confocal microscopy:
- Advantages: out of focus light eliminated by pinhole and consecutive scanning can improve
resolution and contrasts
- Disadvantages: time resolution is limited by scanning speed and short wavelength excitation
can be toxic

79. Imagine you are a PhD student in a Tübingen neuroscience lab and plan an experiment to image
the live transport of fluorescence protein labels GABA receptors from the nucleus to distal
dendrites. For simplicity you culture the transfected neurons in a monolayer culture dish. Name
one imaging method with two advantages and two disadvantages.

80. A neuroscientist in Heidelberg wants to perform time-laps imaging of transport processes of

proteins/protein clusters along the cytoskeleton in living glial cells. The proteins of interest are
flagged/linked to a fluorescence protein that can be directly imaged. Name two different
 microscopy methods that could be used and one advantage and one disadvantage of each
chosen method.

Confocal microscopy:
- Advantages: out of focus light eliminated by pinhole and consecutive scanning can improve
resolution and contrasts compared to regular fluorescence
- Disadvantages: time resolution is limited by scanning speed and short wavelength excitation
can be toxic
2-photon microscopy
- Advantages: infrared light for excitation  no phototoxicity (important to consider given the
fact that a time lapse is performed
- Disadvantage  Limited temporal resolution