We present a simple and robust way to reject out-of-focus background when performing deep two-photon excited fluorescence (TPEF) imaging in thick tissue. The technique is based on the use of a deformable mirror (DM) to introduce illumination aberrations that preferentially degrade TPEF signal while leaving TPEF background relatively unchanged. A subtraction of aberrated from unaberrated images leads to background rejection. We present a heuristic description of our technique, which we corroborate with experiment. Images of a labeled mouse olfactory bulb are compared with standard TPEF microscopy images, demonstrating significant out of focus TPEF background rejection with our technique. Finally we improve our technique by developing a faster aberration modulation mechanism that performs background subtraction line by line rather than frame by frame. In this manner, the overall image acquisition rate of our technique is the same as that of a standard TPEF microscope.

Date Published: 9 February 2007
PDF: 10 pages
Proc. SPIE 6467, MEMS Adaptive Optics, 646704 (9 February 2007); doi: 10.1117/12.707443

Published in SPIE Proceedings Vol. 6467:
MEMS Adaptive Optics
Scot S. Olivier; Thomas G. Bifano; Joel A. Kubby, Editor(s)