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Simulation of two-photon scanning microscopy

Finding the right combination of a probe and a laser for a desired class of biological samples is a defining step in microscopy. As lasers prices vary significantly, it is of a particular importance to choose the best laser for the money without substantial loss in resolution.

In this note, we show how to pre-test lasers, when looking for an alternative to a usually expensive Ti:Sapphire laser, by performing SimphoSOFT® numerical calculations. We calculated the number of fluorescent photons generated from a given set of probes for a series of lasers – Spectra Physics MaiTai, Coherent Chameleon Ultra I, and Toptica Photonics FemtoFErb 780 – to see their performance in two-photon microscopy. Below is one sample from the results acquired for Alexa 488 probe. The results indicate one can use a cheaper fiber laser to reach approximately the same image quality as in the case of a typical expensive Ti:Sapphire. Check out the last column to compare quantitatively the fluorescence signals from the Alexa 488 probe predicted by SimphoSOFT® for different excitation lasers.

Excitation laser
(Alexa 488)
Laser pulse
laser power
Laser pulse
Fluorescence output
(# photons per 100 pulses)
Toptica FemtoFErb 780 (fiber) 780 100 10 0.10 92,500
Spectra Physics MaiTai BB (Ti:Sapphire) 780 80 10 0.125 179,600
Coherent Chameleon Ultra (Ti:Sapphire) 780 140 10 0.125 103,200

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