2-Photon Functional Imaging

Question 1

What does a femtosecond laser do?

Answer 1

Ultra-short pulsed laser -

Question 2

Outline the anatomy of a 2-photon microscope.

Question 3

2-photon fluorescence can image structures in the living brain up to what depth?

Answer 3

1mm

Question 4

Name a molecule that is autofluorescent.

Answer 4

NADH

Question 5

Describe 2 methods for making the brain fluorescent that utilise a dye.

Answer 5

1. Single-cell loading: Alexa-594
2. ‘Bolus’ loading extracellular space: Oregon Green BAPTA (OGB)

Question 6

How can astrocytes alone be visualised by 2-photon microscopy?

Answer 6

Add a dye: Astrocyte label SR101 “dropped” onto cortex

Question 7

How can vasculature of the brain be visualised?

Answer 7

Fluorescent dextran injection reveals vasculature

Question 8

How do we detect neural firing in 2P-M?

Answer 8

• Calcium detection
• Voltage detection

Question 9

Describe the advantages and disadvantages of calcium fluorescence using dyes.

Answer 9

Advantages:

• Quick and easy to label neurons
• Fluorescence increases linearly with [Ca²⁺]
• High sensitivity dyes

Disadvantages:

• Cannot label specific neurons
• Only suitable for acute imaging - on a single day

Question 10

Describe the advantages and disadvantages of using calcium fluoresence with genetically encoded calcium indicators (GECI) - e.g. GcAMP protein family.

Answer 10

Advantages:

• Can label specific neurons - viral injection, cre/lox
• Can be used for chronic imaging

Disadvantages:

• Time consuming - viral protein expression, animal breeding
• Fluorescence does not increase linearly with [Ca²⁺]
• Low sensitivity

Question 11

Using a lower energy (longer wavelength) of light to excite photons results in the emission of red rather than green light. What is the advantage of doing this?

Answer 11

Using lower energy allows for deeper penetration into the tissue and better resolution at an equivalent depth compared to higher energy light.

Question 12

Why is the protein class of genetically encoded voltage indicators - e.g. FlaSh - not currently very useful in 2P-M?

Answer 12

• Sensitivity is very low
• Hard to visualise emissions

Question 13

Why does single photon microscopy produce images with lower resolution than 2-photon microscopy?

Answer 13

In single photon microscopy photons are absorbed in multiple planes, therefore the visualisation is less precise.

Question 14

If I want to image neurons firing in layer 4 of the cortex (400um deep), which of the following should I use?

A) Confocal microscopy and GFO

B) 2 photon microscopy and GFP

C) 2 photon microscopy and GcAMP

D) 2 photon microscopy and FlaSh

Answer 14

C) 2 photon microscopy and GcAMP - using a genetically encoded calcium indicator with 2 photon microscopy it is possible to image the ongoing activity of the neurons, with potentially better visualisation than with FlaSh.

Using GFP would only enable a static image, and would not show the neurons firing action potentials.