UC San Diego

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 η, 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±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±0.01) and 2p excitation efficiency (0.86±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.