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J Neurosci Methods. 2011 Jun 15;198(2):172-80. doi: 10.1016/j.jneumeth.2011.03.015. Epub 2011 Mar 30.

Efficient large core fiber-based detection for multi-channel two-photon fluorescence microscopy and spectral unmixing.

Author information

1
INSERM, U603, Paris F-75006, France.

Erratum in

  • J Neurosci Methods. 2011 Aug 30;200(1):94. van't Hoff, Marcel [corrected to van 't Hoff, Marcel].

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.

PMID:
21458489
DOI:
10.1016/j.jneumeth.2011.03.015
[Indexed for MEDLINE]

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