Compact simultaneous label-free autofluorescence multi-harmonic microscopy for user-friendly photodamage-monitored imaging.

SIGNIFICANCE

Label-free nonlinear optical microscopy has become a powerful tool for biomedical research. However, the possible photodamage risk hinders further clinical applications.

AIM

To reduce these adverse effects, we constructed a new platform of simultaneous label-free autofluorescence multi-harmonic (SLAM) microscopy, featuring four-channel multimodal imaging, inline photodamage monitoring, and pulse repetition-rate tuning.

APPROACH

Using a large-core birefringent photonic crystal fiber for spectral broadening and a prism compressor for pulse pre-chirping, this system allows users to independently adjust pulse width, repetition rate, and energy, which is useful for optimizing imaging conditions towards no/minimal photodamage.

RESULTS

It demonstrates label-free multichannel imaging at one excitation pulse per image pixel and thus paves the way for improving the imaging speed by a faster optical scanner with a low risk of nonlinear photodamage. Moreover, the system grants users the flexibility to autonomously fine-tune repetition rate, pulse width, and average power, free from interference, ensuring the discovery of optimal imaging conditions with high SNR and minimal phototoxicity across various applications.

CONCLUSIONS

The combination of a stable laser source, independently tunable ultrashort pulse, photodamage monitoring features, and a compact design makes this new system a robust, powerful, and user-friendly imaging platform.