SIM and Light Sheet Microscopy

Question 1

SIM

Answer 1

Structured Illumination Microscopy

Image with shiftied periodic stripe patterns. Then if there are two PSF emitters very close to eachother the pattern will predominantly illuminate one of the emitters. Then as the pattern moves it will predominantly illuminate the other. THus when you combine these images you will get a better resolution.

SIM is a product of the Moire effect when undersampling.

So then the OTF is essentially extended 2x.

Question 2

2D SIM considerations

Answer 2

the 2D stripe pattern needs to be shifted
3 times
rotated 3 times (-k0, 0, +k0 where k0 is the Nyquist frequency)
= 9 images in total needed. So then data redundancy is needed.

In addition artifacts from the reconstruction could appear due to imperfect disentanglement of the 9 images. (phase shifts cannot be perfectly precise) howevrer they are easily recognizeable as they will be hexagonally symmetric

Question 3

3D SIM

Answer 3

Needs:
* more illumination patterns (3 angles x 5 shifts/focal slice)
* 2X better in-plane resolution
* better contrast by optical sectioning
* processing bit more involved

Question 4

Practical’s on SIM

Answer 4

Offers 2x increased resolution with good signal transfer of all
spatial frequencies.
* Works in 3D! Can increase axial resolution too. Only need
more patterns in 3D.
* Compatible with any fluorophore/labeling, no special
biochemistry needed!
* To make it work, there are many practical problems:
* projected pattern positions on the sample must be known with
nm accuracy
* multiple acquisitions with different pattern positions must be
acquired
* the digital reconstruction adds “structured noise”

Question 5

Light Sheet Microscopy

Answer 5

Illuminates only plane of interests with a sheet of light. Thus light damage is minimized.

Super simple, but (re)invented by Ernst Stelzer ~2004,
first documented 1903!
* Not very high resolution, but unlimited imaging time!
* Uses very low illumination dose, compatible with outside
sunshine
* Imaging of whole embryos for days possible
* Biggest hit in developmental biology
* Opto-mechanical setup can be complex

Question 6

Ways of producing a light-sheet

Answer 6

The sheet of light is typically
obtained by scanning a laser line very fast up and down. However this requires a very specific illumination setup perpendicular to the objective

Question 7

Lattice Light Sheet

Answer 7

SIM & LightSheet together got a baby

Question 8

Single objective Light Sheets

Answer 8

using a 45deg mirror in the a sample so the illumination sheet can still pass perpeondicluarly through the sample. But ofc this is also not very easy.

or

Oblique Plain Microscopy: use a diagonal light sheet on the same objective. But the to get an image requires a more complex optical setup.
https://andrewgyork.github.io/high_na_single_objective_lightsheet/