Confocal Microscopy Flashcards
General principle of fluorescence microscopy
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
The most crucial phenomenon for fluorescen microscopy
Stokes shift: emitted photon by a fluorophore has a higher wavelength than excitation/absorption photon
Epifluorescence microscope
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
name of mirrors that reflect light only at certain angles
dichroic mirror
Main advantage of inverted microscpes over upright ones
More space available to put sample on top
while the imaging setup is on the bottom
Types of fluorophores
- small synthetic organic molecules
- VFPs
How come VFPs can be fluorescent
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.
typical duty cycle of fluorophore
1-5ns => max 10^9 photons/s
How to ensure high enough signal and why is that a problem in the first place?
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.
How do you quantify fluorescnece efficiency of fluorophores?
- Extinction coefficient: how likely is a fluorophore to absorbs photon
- Quantum yield: fraction of excited molecules which emit a photon
Typical values of quantum yield
0.15: Cy3
0.3: Cy5
needs to be >0.1
Typical values of extinction coefficient
136k: Cy3
250k: Cy5
needs to be above 10k [1/(M*cm)]
Absorption formula
-log_10 (I/I0) = extinction_coeff x pathlength x concentration
Formula for quantum yield
k rad/(k unrad + k rad), it is a probability
How many photons and what power would you generally need to get good amount of emision photons
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
What are the sources of photon signal loss?
- The fluorescence yield of the fluoropohore
- Low laser power
- High NA objective (gets ~5-10% of photons)
- Camera (90-95% efficiency)
- Light cut-off for noise reduction
Sources of fluorescence noise
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)
the main problem confocal microscopy solves
getting sharp 2D images of 3D objects without getting hazyness due to light from other unfocused layers.
Widefield microscopy vs Scanning microscopy
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
Rescan confocal
- 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.
General principle of STED
- 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”
Some setup consideration for STED
- 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
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.
B
What is the power needed for depletion laser?
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.
Abbe’s scriterion modification for STED
in the denominator *sqrt(1+Isted/I)
FRET
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
