Efficient Fiber-Coupled Detection for Multiphoton Microscopy: Characterization and Comparison with Air-Coupled Detection

摘要

The femtoliter excitation volume of multiphoton microscopy renders all emitted photons useful in detecting fluorescence signals. We have previously demonstrated the viability of transmitting the emitted fluorescence via multimode optical fibers onto a photomultiplier tube (PMT) in a full-field arrangement. A custom MPLSM, based on a commercially available confocal microscope was developed to readily switch between the regular descanned path for confocal microscopy and our non-descanned pathways (direct detection) supporting fiber-coupled detection. We now wish to demonstrate the efficacy of fiber-based detection using a side-by-side comparison of our fiber-coupled paradigm to the traditional method of directly focusing the fluorescence, through air, onto a PMT. To effectively compare the two methods, we have incorporated a second direct detection epi-fluorescence pathway for air-coupling onto a PMT that does not affect the performance of the fiber-based MPLSM. We found that fiber-based detection compares favorably against traditional direct detection. We demonstrate the viability of fiber-based detection for high-resolution neuronal brain slice imaging.