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

摘要

Here we present the first results of a new multi photon excitation microscopy setup which extends the excitation wavelengths far beyond one micron. A synchronously pumped femtosecond-OPO (OPO PP-Automatic, APE) pumped by a femtosecond-Ti:Sapphire oscillator is used as the light source. Up to 500nm tuning can be achieved in the NIR (for instance 1100-1600nm) with fixed pump frequency and a single optics set. Automated tuning makes it an ideal tool for MPE-, SHG- and THG microscopy, which is demonstrated in combination with an optimized scanner / microscope / detection system. Together with the Ti:Sapphire pump laser (Coherent Chameleon) excitation wavelengths from 700 to 1600nm are achieved. A single-beam scanner (LaVision BioTec) was optimized for Ti:Sapphire and OPO wavelength ranges including dispersion compensation for maintaining the short pulses at the sample site as well as an overall transmission of 30-38% for the OPO range (measured up to 1400nm). Measurements on human dermis with excitation above 1 micron, compared to lower wavelengths, showed doubling of the penetration depths, strongly reduced photo damage, and by 30fold increased excitation efficiency and 10fold reduced photobleaching of red fluorescent dyes, including RFP and Cy5.5. 1100nm-excited SHG efficiency of collagen was at the same level, compared to 880 nm, yet autofluorescence was decreased by up to 30 times resulting in a significantly improved signal-to-noise ratio for the detection of fluorescent dyes. The resolution is slightly reduced in comparison to Ti:Sapphire excitation, which corresponds well to the longer excitation wavelength used.