2-photon imaging

Question 1

What is 2-photon imaging

Answer 1

• enables high resolution, fluorescence imaging in deep tissue
• use 2 photons to excite sample - they are of lower energy so can penetrate deeper into tissue, however need 2 to excite sample
• gives single point where the 2 photons meet

Question 2

Single photon confocal microscopy

Answer 2

• sample on a slide
• looking down at it, bouncing light down, which reflects straight back up to objective
• laser sends down photons onto the sample, excites it, electrons jump up a shell, return to ground state and emit photon of light with longer wavelength

Question 3

What are the components of a 2-photon microscope?

Answer 3

• pulsed laser - only fire at intervals, power up and fire denser photons. gives increased likelihood that 2 photons will hit
• moving mirrors that deflect light around the axes to hit all the important points
• scan lens and tube lens determine how far the scanning mirrors can move
• Dichoric mirror allows excitation photons through, but deflects emission photons
• PMT collects emitted photons - amplifies areas of signal and dampens areas where there isnt - increases contrast

Question 4

Methods for making the brain fluoresce

Answer 4

• Do nothing - NADH is autofluorescent (if not studying metab., there wont be much though)
• Add a dye - label single neuron/astrocytes/ vasculature/EC space with salt load
• Genetic methods - Use GFPs in viral injections or CreLox system

Question 5

Looking at brain function

Answer 5

• Calcium detection (synaptic transmission)

- Voltage detection (APs)

Question 6

Making calcium fluoresce - DYES

Answer 6

• FURA-2, when binds calcium, it has a change in structure causing it to fluoresce
• very quick and easy to dye the neurons
• fluorescence increases linearly with increase in [Ca]
• highly sensitive
• HOWEVER - cannot label specific neurons and only suitable for acute imaging as dye is metabolised

Question 7

Making calcium fluoresce - Alternative

Answer 7

• gentically ecoded calcium indicator proteins (GCaMP)
• can label specific neurons (viral, Cre/lox)
• can be used for chronic imaging
• HOWEVER - time consuming (takes time for breeding/viral gene expression). Fluorescence increases NON-linearly with [Ca]. Low sensitivity.

Question 8

Green vs Red lasers

Answer 8

• Atm we use green laser, new ones are going to be more towards red end of spectrum
• allows us to see deeper into tissue without having to use high powered laser (burns stuff)

Question 9

How can we sense voltage?

Answer 9

• genetically encode voltage indicator proteins such as FlaSh
• not yet great at reporting AP
• needs a lot of work