Super resolution microscopy methods

Question 1

Abbie´s law, PSF and super resolution methods

Answer 1

Point Spread Function: smallest volume that you can observe from an optical system from fluorescence light microscopy (x- 250nm and z - 500nm)
Super resolution - circunvent this diffraction limit

Question 2

choise based on

Answer 2

acquire data
resolution
thickness
size
damage
speed
context

Question 3

types

Answer 3

Stimulated emission depletion (STED)
Single molecule localization (SMLM) - PALM or STORM
Structure stimulation (SIM)

Question 4

SIM, application and Fourier transform of PSF

Answer 4

sample excited with patterned illumination highlighting - moire effect/fringes (light/dark bands seen by superimposing 2 nearly identical arrays of lines or dots)
unknown sample multiplied by known regular illumination pattern - unobservable information is deduced and computionally restored
3 diffraction orders placed at the limit of backfocal plane of lines
+: speed
-: not much resolution

spatial statistics, intensities, volumes, relative distributions, changes in disease states

Fourier transform: observable region of reciprocal space less resolution information - moire fringes give + 2 offset regions

Question 5

SMLM and new DNA paint

Answer 5

fluorophores randomly activated, imaged and bleached switched to dark state - movies of blinking
information about their location - coordinates- map
-: O2 scavanger systems, slow, not too many at the same time, resolution depends of number of localised molecules

STORM: localise frame by frame and acquire super resolution point

PALM: dark non activated protein that only activates with fluorescence and you can track it (less background signal)

Cluster analysis and quantification - specific patterns about the proteins distribution

DNA paint: Ab with peptide oligo (docking strand) detected by immature strand where hybridization bounds to fluorophore (less toxicity and background)

Question 6

STED

Answer 6

targeted PSF engineered effectively shrinks illumintation spot, just receives fluorescence from central spot (excitation light and depletion laser)
more resolution - more intensity of depletion - - PSF
doesnt require computation to reconstruct data

Question 7

MINI FLUX

Answer 7

STORM + STED - blinking surface with excitation beam in torus (lights around the centre)
more time and toxic
point scanner CDCs like confocal

Question 8

srCryoCLEM

Answer 8

Cryogenic correlative light and EM
1- image with super resolution to receive fluorescence image
2- EM

Question 9

how to choose? triangle

Answer 9

spatial resolution
depth
temporal resolution/speed
photodamge/bleaching