Optimization of multiphoton excitation microscopy by total emission detection using a parabolic light reflector |
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Authors: | CHRISTIAN A COMBS ALEKSANDR V SMIRNOV† JASON D RILEY‡ AMIR H GANDJBAKHCHE‡ JAY R KNUTSON† & ROBERT S BALABAN§ |
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Affiliation: | NHLBI Light Microscopy Facility;NHLBI Laboratory of Molecular Biophysics;, NICHD Laboratory of Integrative and Medical Biophysics and the;NHLBI Laboratory of Cardiac Energetics, National Institutes of Health, Bethesda, MD 20892-1061, U.S.A. |
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Abstract: | We have constructed a device that maximizes the probability of collecting all of the scattered and ballistic light isotropically generated at the focal spot of multiphoton excited emissions (MPE) to optimize the signal-to-noise ratio (SNR) for micro-imaging. This was accomplished by optically coupling a parabolic reflector (that surrounds the sample and top of the objective) to a pair of collimating lenses (above the sample) that redirects emitted light to a separate detector. These additional optics, combined with the objective, allow the total emission detection (TED) condition to be approached. Numerical simulations suggest an approximately 10-fold improvement in SNR with TED. Comparisons between the objective detection and TED reveal an enhancement of 8.9 in SNR (77% of predicted) for GFP-labelled brain slices and similar results for fluorescent beads. This increase in SNR can be used to improve time resolution, reduce laser power requirements/photodynamic damage, and, in certain cases, detection depth, for MPE imaging techniques. |
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Keywords: | Brain GFP light collection trans-fluorescence two-photon microscopy |
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