
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 type |
Excitation wavelength (Alexa 488) |
Laser pulse length (fs) |
Average laser power (mW) |
Laser pulse energy (nJ) |
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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SimphoSOFT® can be purchased as a single program and can be also configured with Energy Transfer add-on ![]() ![]() ![]() ![]() ![]()
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