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Optimization of multiphoton excitation microscopy by total emission detection using a parabolic light reflector.

Combs, Christian A; Smirnov, Aleksandr V; Riley, Jason D; Gandjbakhche, Amir H; Knutson, Jay R; Balaban, Robert S.
J Microsc; 228(Pt 3): 330-7, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18045327
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.
Selo DaSilva