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

ABSTRACT:

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.

SUBMITTER: Saidi S 

PROVIDER: S-EPMC10704185 | biostudies-literature | 2023 Oct

REPOSITORIES: biostudies-literature