Wavelength dependent damage in biological multi-photon microscopy: Ti:sapphire vs. Cr:forsterite lasers

We report our study of wavelength dependent on-focus cell damage in multi-photon confocal microscopy with femtosecond IR laser sources. Experiments were performed in selected plant tissues. Two different laser sources were selected: a femtosecond Ti:sapphire laser and a femtosecond Cr:forsterite laser. We selected Cr:forsterite laser as the comparison laser source due to its emission wavelength (1230 nm in this study) longer than the three-photon absorption threshold of protein. Similar to previous studies, with more than 10 mW micro-irradiation at 800 nm from a femtosecond Ti:sapphire laser, multi-photon absorption induced cell damages due to plasma generation, accompanied with strong luminescence, can be frequently observed in our studies. The dosages (illumination time) required to produced plasma generation (characterized by strong luminescence) were found to decrease with increased illumination intensity. In contrast, with a longer output wavelength from a Cr:forsterite laser at 1230 nm, multi-photon absorption and auto-fluorescence were found to be significantly suppressed and the cell damage was found to be greatly reduced. Sustained multi-photon spectra can be observed in most plant specimens even with a tightly focused Cr:forsterite laser beam, with similar or higher intensity enough to produce plasma generation at 800 nm wavelength (with over 100 mW average power), under long term irradiation.