Super Resolution Microscopy

Question 1

What is challenging about imaging peroxisomes?

Answer 1

Peroxisomes will be in different focal planes so will not be images

Question 2

What is Z-stacking?

Answer 2

Multiple images across focal planes images which can then be integrated into a 2D image

Question 3

What is a convoluted image?

Answer 3

When out of focus light is generated and reduced resolution

Question 4

What is deconvolution?

Answer 4

A computational technique that removes out of focus light and increases resolution

Question 5

What is at the abbe diffraction liimit?

Answer 5

Light cannot be focused onto an area smaller than half the wavelength of the light itself

Question 6

What does the abbe diffraction limit mean for wide field microscopy?

Answer 6

Determines the minimum size of an object that can be resolved
Also means that two separate fluorescent signals cannot be resolved if the distance between them is less than the wavelength of light emitting them

Question 7

What are the two main types of super resolution microscopy?

Answer 7

Pattern illumination

Single molecule imaging

Question 8

What does pattern illuminating SRM use?

Answer 8

spatial activation of fluorescent molecules in an area - not all fluorophores are excited
Then image multiple different activations and reconstruct into an image
- could be used for peroxisomes

Question 9

What does single molecule SRM use?

Answer 9

Uses photo switchable fluorescent probes which are selectively excited imaged and deactivated - can then reconstruct the image