Optimization of multiphoton excitation microscopy by total emission detection using a parabolic light reflector.
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.
NHLBI Light Microscopy Facility, National Institutes of Health, Bethesda, MD 20892-1061, USA. combsc@nih.gov
Christian A
CA
Combs
Aleksandr V
AV
Smirnov
Jason D
JD
Riley
Amir H
AH
Gandjbakhche
Jay R
JR
Knutson
Robert S
RS
Balaban