Nonlinear excitation fluorescence microscopy: source considerations for biological applications

David L. Wokosin

doi:10.1117/12.773610

14 February 2008 Nonlinear excitation fluorescence microscopy: source considerations for biological applications

David L. Wokosin

Proceedings Volume 6871, Solid State Lasers XVII: Technology and Devices; 687128 (2008) https://doi.org/10.1117/12.773610
Event: Lasers and Applications in Science and Engineering, 2008, San Jose, California, United States

Abstract

Ultra-short-pulse solid-state laser sources have improved contrast within fluorescence imaging and also opened new windows of investigation in biological imaging applications. Additionally, the pulsed illumination enables harmonic scattering microscopy which yields intrinsic structure, symmetry and contrast from viable embryos, cells and tissues. Numerous human diseases are being investigated by the combination of (more) intact dynamic tissue imaging of cellular function with gene-targeted specificity and electrophysiology context. The major limitation to more widespread use of multi-photon microscopy has been the complete system cost and added complexity above and beyond commercial camera and confocal systems. The current status of all-solid-state ultrafast lasers as excitation sources will be reviewed since these lasers offer tremendous potential for affordable, reliable, "turnkey" multiphoton imaging systems. This effort highlights the single box laser systems currently commercially available, with defined suggestions for the ranges for individual laser parameters as derived from a biological and fluorophore limited perspective. The standard two-photon dose is defined by 800nm, 10mW, 200fs, and 80Mhz - at the sample plane for tissue culture cells, i.e. after the full scanning microscope system. Selected application-derived excitation wavelengths are well represented by 700nm, 780nm, ~830nm, ~960nm, 1050nm, and 1250nm. Many of the one-box lasers have fixed or very limited excitation wavelengths available, so the lasers will be lumped near 780nm, 800nm, 900nm, 1050nm, and 1250nm. The following laser parameter ranges are discussed: average power from 200mW to 2W, pulse duration from 70fs to 700fs, pulse repetition rate from 20MHz to 200MHz, with the laser output linearly polarized with an extinction ratio at least 100:1.

Citation Download Citation

David L. Wokosin "Nonlinear excitation fluorescence microscopy: source considerations for biological applications", Proc. SPIE 6871, Solid State Lasers XVII: Technology and Devices, 687128 (14 February 2008); https://doi.org/10.1117/12.773610

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