Final Answer

Final question of my last final of the semester:

Calculate the total fluorescence generated per second in the two photon

excitation volume created using a high NA objective lens. 

The parameters are:

Concentration C = 1 μM Fluorescein

Excitation wavelength = 800 nm

2P action cross-section σ2p = 34 GM

Femtosecond laser intensity = 1 mW

Laser repetition rate = 80 MHz

Laser pulse width = 100 fs

Lens NA (water immersion) = 1.2, n = 1.33

Other info: The lens back aperture is overfilled so there is a fully diffraction-limited focus and you can assume that the focal volume (PSF) is a 3D Gaussian function. The action cross-section includes the quantum yield so you don’t need to consider it in the calculation. Remember that the intensity you use in the calculation will have dimensions of photons per area per second and the value is that of the center of the PSF, in other words I(0,0,0). This intensity should be normalized to the area at z = 0 for the one photon PSF and then squared. I(0,0,0)2 will be multiplied by the focal volume equation to calculate the total number of excitations per second. You can neglect any attenuation of the light as it passes through the focus.

Final Answer: 9.5 x 10⁶ excitations/s

I'm done with my first year of graduate school!!!!