Dr. Wafa Amir Profile

Dr. Wafa Amir

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Proceedings Article | 15 October 2007 Paper

Simultaneous imaging of multiple focal planes in scanning two-photon absorption microscope by photon counting

Ramón Carriles, Erich Hoover, Wafa Amir, Jeffery Squier

Proceedings Volume 6771, 67710C (2007) https://doi.org/10.1117/12.731868

KEYWORDS: Clocks, Microscopes, Luminescence, Field programmable gate arrays, Photon counting, Objectives, Control systems, Electronics, Beam splitters, Data transmission

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We demonstrate a two-photon absorption scanning microscope capable of imaging two focal planes simultaneously. The 23MHz fundamental laser is split in two, one part delayed in time while the other is focused with a deformable mirror to change its divergence. Both parts are then recombined to form a 46MHz pulse train consisting of two interlaced trains with different divergences that after the objective are focused at different sample depths. At the detection path, photon counting techniques allow photons coming from each depth to be separated based on their relative timing with respect to the 46MHz train. The separation is accomplished using a field-programmable gate array that has been programmed to switch back and forth between two counters at a rate provided by a master clock generated by the 46MHz pulse train. The computer that controls the scanners reads and resets the counters before moving to a new pixel. The scheme is demonstrated for two depths but can be extended to a larger number, the ultimate limit being the fluorescence lifetime. This technique could also be implemented for second or third harmonic generation microscopy, in this case the ultimate achievable number of focal planes would be determined by the electronics speed.

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