Evaluation of compact pulsed lasers for two-photon microscopy using a simple method for measuring two-photon excitation efficiency

Significance: Two-photon (2p) microscopy has historically relied on titanium sapphire pulsed lasers that are expensive and have a large footprint. Recently, several manufacturers have developed less expensive compact pulsed lasers optimized for 2p excitation of green fluorophores. However, quantitative evaluation of their quality is lacking. Aim: We describe a simple approach to systematically evaluate 2p excitation efficiency, an empiric measure of the quality of a pulsed laser and its ability to elicit 2p induced fluorescence. Approach: By measuring pulse width, repetition rate, and fluorescence output, we calculated a measure of 2p excitation efficiency., which we compared for four commercially available compact pulsed lasers in the 920 to 930 nm wavelength range. Results: 2p excitation efficiency varied substantially among tested lasers. The Coherent Axon exhibited the best 2p excitation efficiency (1.09 similar to 0.03), exceeding that of a titanium sapphire reference laser (defined to have efficiency = 1). However, its measured fluorescence was modest due to its long pulse width. Of the compact lasers, the Toptica Femtofiber Ultra exhibited the best combination of measured fluorescence (0.75 similar to 0.01) and 2p excitation efficiency (0.86 similar to 0.01). Conclusions: We describe a simple method that both laser developers and end users can use to benchmark the 2p excitation efficiency of lasers used for 2p microscopy.