Optimizing Multi-Photon Fluorescence Microscopy Light Collection from Living Tissue by Non-Contact Total Emission Detection (TEDII)

Abstract

A benefit of multiphoton fluorescence microscopy is the inherent optical sectioning that occurs during excitation at the diffraction-limited spot. The scanned collection of fluorescence emission is incoherent; i.e., no real image needs to be formed on the detector plane. The isotropic emission of fluorescence excited at the focal spot allows for new detection schemes that efficiently funnel all attainable photons to detector(s). We previously showed (JOM v.228, p.330-7, 2007) that parabolic mirrors and condensers could be combined to collect the totality of solid angle around the spot for tissue blocks, leading to ∼8-fold signal gain. We now apply a version of this Total Emission Detection instrument modified to make non-contact images inside tissue in vivo. The device is mounted on a periscope (LSM Tech) to avoid touching tissue and is simpler, and in some cases more effective, than hybrid objective and fiber optic ring based systems for emission collection enhancement. Images of live brain and kidney show that the gain using this optical scheme varies as a function of imaging depth and the characteristics of the sample being imaged. Brain imaging (through a tiny region of thinned skull) of GFP labeled microglia showed up to a 1.8 fold increase in emission collection, while the gain in whole ex vivo brain samples showed up to a 2.5 fold increase (vs. light collected by a 20X water 0.95NA lens alone). Rat kidney imaging of blood vessels labeled with anneps (Invitrogen) in vivo showed up to a 2 fold enhancement in emission collection. These results show that multi-photon imaging using the TEDII device will permit scanning at twice the rate with the same SNR in these tissues or allow reduction of laser power by 60% to reduce photo-damage.