2 photon imaging

Question 1

What’s the difference between 1p and 2p imaging?

Answer 1

1p:
- high scattering of axciting photons
- high absorbance of exciting photons
- linear, large volume emission

2p:
- low scattering of exciting photons –> good signal:noise ration –> deeper imaging
- low absorbance of exciting photons –> less laser power needed –> less damage
- non-linear, point-like emission –> no need for a pinhole/descan –> no out of focus light

Question 2

What is 2p imaging?

Answer 2

Fluorescence imaging with a twist:
2 p with a small delay sent to the fluorophore

Question 3

What are the limitations of 2p microscopy?

Answer 3

• Image depth: up to 800 microm - deeper brain regions are out of reach
• the optical resolution (diffraction limit)
• fluorescent probes: when fluorescent tagging changes the properties of an investigated molecule, or is toxic