Confocal Microscopy

Question 1

General principle of fluorescence microscopy

Answer 1

You excite fluorophore with a specific light wavelength from ground to excited state with light and as it goes back to ground state it emits another wavelnght of light

Question 2

The most crucial phenomenon for fluorescen microscopy

Answer 2

Stokes shift: emitted photon by a fluorophore has a higher wavelength than excitation/absorption photon

Question 3

Epifluorescence microscope

Answer 3

illumination and imaging are from the same side.
This is achieved using dichroic mirror which reflects the excitation light from the laser/light source with filter but then passes the emmited light by the sample as it is at the right angle

Question 4

name of mirrors that reflect light only at certain angles

Answer 4

dichroic mirror

Question 5

Main advantage of inverted microscpes over upright ones

Answer 5

More space available to put sample on top
while the imaging setup is on the bottom

Question 6

Types of fluorophores

Answer 6

1. small synthetic organic molecules
2. VFPs

Question 7

How come VFPs can be fluorescent

Answer 7

They have a beta barrel structure which encapsulates 3 conserved amino-acids, Ser-Tyr-Gly. The tyr is fluorescent as it has an aromatic ring. However, the local environment defined by the protein structure around these aas determines the wavelength properties.

Question 8

typical duty cycle of fluorophore

Answer 8

1-5ns => max 10^9 photons/s

Question 9

How to ensure high enough signal and why is that a problem in the first place?

Answer 9

Fluorescence of fluorophores occurs at a low probability. Thus ensuring you have good quality fluorophores with the correc wavelenght properties for your laser setup and having high power lasers is crucial to ensure high enough fluorescent signal.

Question 10

How do you quantify fluorescnece efficiency of fluorophores?

Answer 10

1. Extinction coefficient: how likely is a fluorophore to absorbs photon
2. Quantum yield: fraction of excited molecules which emit a photon

Question 11

Typical values of quantum yield

Answer 11

0.15: Cy3
0.3: Cy5

needs to be >0.1

Question 12

Typical values of extinction coefficient

Answer 12

136k: Cy3
250k: Cy5

needs to be above 10k [1/(M*cm)]

Question 13

Absorption formula

Answer 13

-log_10 (I/I0) = extinction_coeff x pathlength x concentration

Question 14

Formula for quantum yield

Answer 14

k rad/(k unrad + k rad), it is a probability

Question 15

How many photons and what power would you generally need to get good amount of emision photons

Answer 15

10^12 photons or 20W
=> 10^5 emited photons
terrible

*generally diode lasers are ~25-100mW but are focused with lenses to achieve higher power

Question 16

What are the sources of photon signal loss?

Answer 16

1. The fluorescence yield of the fluoropohore
2. Low laser power
3. High NA objective (gets ~5-10% of photons)
4. Camera (90-95% efficiency)
5. Light cut-off for noise reduction

Question 17

Sources of fluorescence noise

Answer 17

Scattering from solvent. Water molecules are 10^-12 less efficient fluorophores but are 10 order more prevalent so they are a problem.

Contamination on sample slide can also have some fluorescent activity. That is why it is important to clean them (with fire and/or KOH)

Question 18

the main problem confocal microscopy solves

Answer 18

getting sharp 2D images of 3D objects without getting hazyness due to light from other unfocused layers.

Question 19

Widefield microscopy vs Scanning microscopy

Answer 19

Widefield microscopy: acquisition of
image points in parallel by imaging onto
pixilated detector
* has uniform illumination
* uses an image sensor for
parallel imaging
* is fast
* has poor optical sectioning

Scanning microscopy: acquisition of
image points sequentially by scanning
focused laser beam relative to sample
* has single spot illumination
* uses a scanner for sequential
imaging
* is slow
* has good optical sectioning
* SNR depends on the size of
the pinhole

Question 20

Rescan confocal

Answer 20

• Available commercially
• Offers increased resolution
• Theoretical 2x better, but limited by strong decay of the OTF
  at high spatial frequencies; practically only ~1.4x better.
• Very easy to retro-fit on existing confocal microscopes.
  After the emitted fluorescence light passes through the confocal pinhole, it is directed onto a second scanning system, called the rescan unit.
  This rescan unit performs a synchronized scan of the detected fluorescence light, effectively “rescanning” the image onto a camera (such as a sensitive CCD or sCMOS camera) at a slightly different rate.
  This rescan step takes the detected signal and spatially oversamples it, effectively increasing the resolution.

Question 21

General principle of STED

Answer 21

• Use scanning microscope
• Excite fluorophores with first spot
• Illuminate with second “doughnut” spot
• Deplete excited state via stimulated emission
• Collect fluorescent light from central “spike”

Question 22

Some setup consideration for STED

Answer 22

• STED-beam must have bit larger wavelength (Stokes-shift)
• STED-spot must be engineered to “doughnut”-beam/ring-shaped spot
• Needs high powers to fully deplete excited state

Question 23

What should one do to get the best
STED resolution?
A. Shift wavelength of depletion laser to the red.
B. Increase the power of depletion laser.
C. Increase the power of the excitation laser.
D. Scan the beams faster.

Answer 23

B

Question 24

What is the power needed for depletion laser?

Answer 24

Probability that photon de-excites fluorophore by stimulated
emission must be very close to one.

photon-hits = intensity X cross-section X excited state lifetime

Thus Laser powers of several W needed!!! otherwise the resulting signal spot will be bigger.

Question 25

Abbe’s scriterion modification for STED

Answer 25

in the denominator *sqrt(1+Isted/I)

Question 26

FRET

Answer 26

Fluorescence/Foerster Resonance Energy Transfer
* Non –radiative transfer of energy of close-by fluorophores
The rate of energy transfer is heavily
dependent on the distance between dyes and thus allows for detecting interactions between molecules. The closer molecules are there will be more fluorescent signal from the acceptor fluorophore(e.g. Cy3) and not the donor(Cy5)

The energy transfer depends on:
* Spectral overlap of donor emission & acceptor
absorption spectra
* Relative orientation of donor & acceptor dipole
orientation
* Quantum yield of the acceptor