SMLM2

Question 1

**

Resolution criteria in super-resolution

Answer 1

Nyquist sampling density:
2 /ρ
* Localization precision:
σ

Resolution depends on
localization precision
and labelling density

Question 2

FRC resolution

Answer 2

Fourier Ring Correlation
tests how well the FT of the two halves of localization time series correlate with eachother. THe threshold frequency below which the correlation drops below a certain value (1/7 for some reason) is the indicator of resolution.

Question 3

How does FRC resolution relate to frame acquisition time?

Answer 3

FRC drops nonlinearly (kind of logarithmically looking) with increasing frame acqusition time , equivalently, with the density of localized labels.

Question 4

stage drift

Answer 4

the unavoidable slight movement of the stage over time. Postprocessing is thus necessary

Question 5

Main challenge in 3D localization microscopy

Answer 5

The PSF is symmetric over the z-axis and changes very slowly. Thus based on its shape it is hard to determine z-location

Question 6

Most common way to localize PSF centre on the z-axis

Answer 6

Introduce astigmatism:
either with cylindrical lenses or dual focal plane (this one splits the FOV and then requires precise overlap registration). Then the PSF shape is shifts significantly and asymetrically along the z-axis providing an indicator for the z-coiordinate

Changing the PSF such that over a range of ~1 μm we can
measure a variation in shape as a function of axial height
* All methods perform about equally well
* Astigmatism method easiest to use
* Axial localization typically 2-4x worse than in plane due to
* depth of focus&raquo_space; lateral spot shape
* PSF footprint on detector larger for detecting shape
(worse signal to background ratio)

Question 7

MINFLUX

Answer 7

Use doughnut beam to illuminate sample n times moving the pot around. This way you excite diferent fluorophores on your speciment which are not in the centre of the doughnut. Not seeing certain fluorophores you have seen in neighbouring illumination tells you they are inside the doughnut.

Provides precision scaling not limited by Abbe but by L/sqrt(N)
(L is the size of the doughnut hole and N the sampling number).

Question 8

advantages and disadvantages of MINFLUX

Answer 8

• Gives better localization precision for same number of photons as
  in traditional localization
• Reason: Include information about the beam position into the
  estimation process! (Prior knowledge is power)
• About 10x less photons needed for same loc. precision
• Allows very fast tracking of one single molecule emitter with very
  high precision

but

very sensitive means very precise control ans very slow.

Question 9

SIMFLUX

Answer 9

widefield version of FLUX.

• only 2 illumination pattern
  directions needed (just wave stripes in x and y)
• at least 3 phases

Thus the same way if the fluorophore is not inside the pattern it will not be excited and will allow for higher resolution

up to ~200 patterns/sec
* ~700 W/cm2 intensity
* 26 μm FOV
(400x400 pixels of 65 nm)

Question 10

Problem: Resolution is still limited because of
* Low density labelling
* Not all labelling sites are visible

What is the way to go for this then?

Answer 10

Solution: Properly combine
many identical copies into a single
reconstruction. Increases the apparent density of labeling

Particle fusion

this allows for subnm resolution

but there is template bias