Super-resolution microscopy

Question 1

Why might super-res be necessary for peroxisomes?

Answer 1

• intracellular position changes as trafficked to different locations w/in cell, so hard to focus on all organelles

Question 2

How can you capture multiple focal planes?

Answer 2

• Z-stacking

- images from range of focal planes integrated to reconstitute single 2D image

Question 3

What is deconvolution?

Answer 3

• computational technique for image processing that increases contrast and resolution of digital images captured on microscope
• achieved by subjecting images to mathematical algorithm that identifies out of focus light and either modifies or removes it from an image

Question 4

What are Widefield microscopes?

Answer 4

• inc brightfield and fluorescent microscopes
• suitable for high-resolution imaging of a diverse range of targets in lab
• but resolution limited by physical properties of light, so not suitable for imaging objects in <1µM range

Question 5

What is the Abbe diffraction limit?

Answer 5

• determines the min size of object that can be resolved by widefield microscopes
• also means that 2 separate
  fluorescent signals cannot be resolved if distance between them is less than
  wavelength of light emitted from them, as 2 light signals will overlap
• many cellular structures smaller than this limit

Question 6

What are the 2 classes of super-res microscopy?

Answer 6

1) uses patterned illumination to differentially and spatially modulate fluorescent behaviour of molecules w/in diffraction-limited region
2) uses single-molecule imaging and photoswitching fluorescent molecules to image individual fluorescent foci

Question 7

How does patterned illuminating super-resolution microscopy (STED/SIM) work?

Answer 7

• rely on the spatial activation of fluorescent molecules w/in defined area
• used to gen image where not all fluorescent molecules in a particular region are excited, so subdiffraction limit resolution is achieved and individual foci can be resolved
• multiple patterns produced across the same sample area and software reconstructs these into a super-resolution image

Question 8

How does single molecule super-resolution microscopy (STORM/PALM) work?

Answer 8

• rely on selective imaging of individual fluorophores, then use series of images to
  reconstitute single high-res image
• need to selectively excite fluorophore in a defined, localised area (not neighbours) and capture emission of single fluorophore
• STORM/PALM use photoswitchable fluorescent probes (switch on/off)
• signals from random
  individual fluorescent foci are far apart, so can undergo digital processing to resolve
  them from other foci and record their location