Neuro 480 Two-Photon Fluorescence Microscopy

Single photon vs Two Photon Excitation

Single Photon - the phtoon has exactly the right energy to jump the fluorophore to the excited state

Two Photon Excitation - happens when a fluorophore asorbs two lower energy photons. Firs photon puts it into a “virtual” state and the seond pushes it into the real excited state which emits fluorescnece. This mimics the effect of single photon but wiht less damage to tissues

What is the two photon cross section?

Probability that a two photon abosrption happens

Units = Goppert-Mayer

Action cross section

Combines absorption probability and quantum eficiency

Tells you how bright a fluorophore will look under two photon excitation

Quantum Efficiency 

Number of photons emitted / number of photons absorbed

What allows the lasers to fire in pulses instead of continious light?

The Ti: Sapphire crystal which works through Kerr lens mode-locking

What is the Kerr effect?

The Kerr effect happens when very intense light changes the way a material bends light (its refractive index). Stronger light causes a bigger change, which can make the material act like it has different optical properties depending on the light’s polarization

In two-photon excitation, why is the excitation rate proportional to P²?

Because two photons must be absorbed simultaneously, making the probability of excitation depend on the square of the photon flux (intensity).

In two photon miceoscopy, what are the two methods used to “crowd” photons together?

Temporally = pulsed lasers pack photons close in time

Spatially = the microscope lens focuses into a very tiny spot

Where can two photon excitation ONLY occur?

ONLY at the focal point of the laser

What is the point of uncaging Ca molecules?

To precisely control the timing and location of intracellular Ca²⁺ increases.

Why is Ca²⁺ uncaging important?

It allows researchers to mimic physiological Ca²⁺ signals (e.g., synaptic plasticity, neurotransmitter release) and study their effects with high spatial and temporal precision.

How does fluorescence collection differ between single-photon confocal and two-photon microscopy?

In single-photon confocal, much of the fluorescence comes from out-of-focus regions and is blocked by a pinhole, so only a small fraction is used. In two-photon microscopy, fluorescence is generated only at the focal plane, so no pinhole is needed and nearly all emitted light can be collected.

What are cons about conofocal imaging?

Photons from outside the focal plan can sometimes sneak through the pinhole - increases background noise 

Scattered photons cant be distinguished form out of focus ones 

• Both wasteful and less clean in scattering tissue 

What does two photon excitation depend on?

Intensity Squared (I²)

What are some caveats/problems that come with two photon microscopy

Risks include:

• photobleaching

• baseline fluorescence artifacts

• protein damage

• abnormal/incorrect Ca signals

• Oxidative stress

What is FRET

FRET (Fluorescence Resonance Energy Transfer) is a technique that detects when two molecules are very close (1–5 nm).

How does FRET indicate protein–protein interactions?

When two proteins fused to different fluorophores (blue + green) come within 1–5 nm, excitation of the donor (blue) transfers energy to the acceptor (green), causing green fluorescence. If proteins don’t interact, only blue light is seen.