Efficient large core fiber-based detection for multi-channel two-photon fluorescence microscopy and spectral unmixing.
Journal of Neuroscience Methods. 2011-06-01; 198(2): 172-180
DOI: 10.1016/j.jneumeth.2011.03.015
Read on PubMed
1. 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.
Ducros M(1), van ‘t Hoff M, Evrard A, Seebacher C, Schmidt EM, Charpak S, Oheim
M.
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].
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.
Copyright © 2011 Elsevier B.V. All rights reserved.
DOI: 10.1016/j.jneumeth.2011.03.015
PMID: 21458489 [Indexed for MEDLINE]