title: Efficient large core fiber-based detection for multi-channel two-photon fluorescence microscopy and spectral unmixing.
abstract: Low-magnification high-numerical aperture objectives maximize the collection efficiency for scattered two-photon excited fluorescence (2PEF), but non-descanned detection schemes for such objectives demand optical components much bigger than standard microscope optics. Fiber coupling offers the possibility of removing bulky multi-channel detectors from the collection site, but coupling and transmission losses are generally believed to outweigh the benefits of optical fibers. We present here two new developments based on large-core fiber-optic fluorescence detection that illustrate clear advantages over conventional air-coupled 2PEF detection schemes. First, with minimal modifications of a commercial microscope, we efficiently couple the output of a 20×/NA0.95 objective to a large-core liquid light guide and we obtain a 7-fold collection gain when imaging astrocytes at 100 μm depth in acute brain slices of adult ALDH1L1-GFP mice. Second, combining 2PEF microscopy and 4-color detection on a custom microscope, mode scrambling inside a 2-mm plastic optical fiber is shown to cancel out the spatially non-uniform spectral sensitivity observed with air-coupled detectors. Spectral unmixing of images of brainbow mice taken with a fiber-coupled detector revealed a uniform color distribution of hippocampal neurons across a large field of view. Thus, fiber coupling improves both the efficiency and the homogeneity of 2PEF collection.
authors: INSERM, U603, Paris F-75006, France.
authors: Mathieu
authors: M
authors: Ducros
authors: Marcel
authors: M
authors: van 't Hoff
authors: Marcel
authors: M
authors: van't Hoff
authors: Alexis
authors: A
authors: Evrard
authors: Christian
authors: C
authors: Seebacher
authors: Elke M
authors: EM
authors: Schmidt
authors: Serge
authors: S
authors: Charpak
authors: Martin
authors: M
authors: Oheim
mesh_terms: D000818:Animals; D001921:Brain / Q000502:physiology*; D004867:Equipment Design; D005336:Fiber Optic Technology; D051379:Mice; D008822:Mice, Transgenic; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D009474:Neurons / Q000502:physiology*; D055100:Optical Fibers