Abstract
We describe a custom one-photon (confocal) and two-photon all-digital (photon counting) laser scanning microscope. The confocal component uses two avalanche photodiodes (APDs) as the fluorescence detector to achieve high sensitivity and to overcome the limited photon counting rate of a single APD ( approximately 5 MHz). The confocal component is approximately nine times more efficient than our commercial confocal microscope (fluorophore fluo 4). Switching from one-photon to two-photon excitation mode (Ti:sapphire laser) is accomplished by moving a single mirror beneath the objective lens. The pulse from the Ti:sapphire laser is 109 fs in duration at the specimen plane, and average power is approximately 5 mW. Two-photon excited fluorescence is detected by a fast photomultiplier tube. With a x63 1.4 NA oil-immersion objective, the resolution of the confocal system is 0.25 microm laterally and 0.52 microm axially. For the two-photon system, the corresponding values are 0.28 and 0.82 microm. The system is advantageous when excitation intensity must be limited, when fluorescence is low, or when thick, scattering specimens are being studied (with two-photon excitation).
Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callMore From: American Journal of Physiology-Heart and Circulatory Physiology
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
