10 July 2003 Fiber-coupled non-descanned 4π detection with a commercial confocal microscope modified for multiphoton imaging
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Abstract
The extremely small (femtoliter) excitation volume of multiphoton (MP) microscopy renders all emitted photons useful in detecting fluorescence signals. Hence, multiphoton laser scanning microscopy (MPLSM) systems can collect fluorescence through the objective (epi-fluorescence), as well as the condenser (trans-fluorescence). For maximal collection efficiency, both optical paths can be used concurrently (4π detection). Most MPLSM systems incorporate photodetectors directly in or adjacent to the epi- and trans-fluorescence optical paths of the microscope, generally photomultiplier tubes with associated optics. These arrangements are optically straightforward, but are often bulky and difficult to reconfigure. Here, we demonstrate that all fluorescence from the specimen can be efficiently coupled into two multimode optical fibers -- one each for the epi- and trans-fluorescence pathways. Fiber-coupled detection enables a modular detection paradigm where light can be routed to easily reconfigurable and interchangeable detection module(s). A novel MPLSM system was constructed, which is readily switched between the original de-scanned detection path for confocal microscopy, and the newly added pathways supporting fiber-coupled non-descanned 4π detection for MP microscopy. Sample MP images of fluorescent beads and fluorescent-labeled hippocampal neurons are presented, demonstrating the viability of fiber-coupled detection.
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Adam M. Larson, Vijay Iyer, Tycho Hoogland, Peter Saggau, "Fiber-coupled non-descanned 4π detection with a commercial confocal microscope modified for multiphoton imaging", Proc. SPIE 4963, Multiphoton Microscopy in the Biomedical Sciences III, (10 July 2003); doi: 10.1117/12.478000; https://doi.org/10.1117/12.478000
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