Infrared multiphoton microscopy beyond 1 micron: system design and biomedical applications

摘要

Here we present a multiphoton excitation microscopy setup extending the excitation wavelengths far beyond one micron. A synchronously pumped fs-OPO (OPO PP-Automatic, APE) pumped by a fs-Ti: Sapphire oscillator is used as the lightrnsource. The biological relevant wavelength range from < 1050 to > 1350 nm can be covered with a fixed pump frequencyrnand a single optics set through hands free, automated tuning. Together with the Ti:Sapphire pump laser (CoherentrnChameleon) excitation wavelengths from 680 to 1600nm are achieved. Two separate scanners (LaVision BioTec) are optimized for Ti: Sapphire and OPO wavelength ranges respectively including dispersion compensation for maintaining the short pulses at the sample site. An overall transmission of 30-38% up to 1400 nm was verified. Measurements on human dermis with excitation above 1 micron, compared to lower wavelengths, showed doubling of the penetration depths, strongly reduced photo damage, a 30fold increased excitation efficiency of red fluorescent dyes, including RFP and Cy5.5. Excitation at 1100 nm further leads to a 4fold decrease in autofluorescence, resulting in a significantly improved signal-to-noise ratio. The resolution is slightly reduced in comparison to Ti:Sapphire excitation, which corresponds well to the longer excitation wavelength used. Phototoxicity and photobleaching is reduced by 80-95%. An OPO pump wavelength around 800nm opens up the possibility to use the Ti:Sapphire laser to pump the OPO and to excite the sample simultaneously giving the opportunity to xcite dyes such as GFP with the pump laser and red shifted fluorophores (for instance RFP) with the OPO at the same time.