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

https://doi.org/10.1016/j.jneumeth.2011.03.015Get rights and content

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.

Highlights

► Fiber-coupled detection optics allows 7-fold better detection of scattered two-photon excited fluorescence on a commercial microscope. ► The full collection power of low-magnification high-numerical aperture objectives is maintained. ► Transmission of optical fibers can be increased to close to 90% by anti-reflex coating of the input and output windows. ► Fiber coupling cancels out spatial variations of the spectral sensitivity on a four-channel detector.

Introduction

Two-photon excitation fluorescence (2PEF) microscopy has become a method of choice for imaging acute brain slices or intact rodent brain, in vivo. Non-linear absorption confines 2PEF to a microscopic isotropic point source, making it possible to collect not only ballistic, but also scattered fluorescence. With increasing imaging depth, the 2PEF signal relies more heavily on the detection of scattered photons (Centonze and White, 1998, Sergeeva, 2010). Strategies to maximize their collection include non-descanned ‘whole-field’ detectors (Denk and Svoboda, 1997), transmitted-light detectors below the sample (Koester et al., 1999, Mainen et al., 1999) or additional epi-detectors around the objective (Combs et al., 2007, Engelbrecht et al., 2009, McMullen et al., 2010, Combs et al., 2011) as well as mirror-coated slides (Rehberg et al., 2010). The purpose of all these strategies is to increase the effective field-of-view and numerical aperture (NA) of fluorescence collection. In the upright recording geometry used in vivo, high-NA objectives having a low magnification (i.e. large field of view) have been shown to offer superior collection efficiency than higher-magnification objectives with similar NA (Oheim et al., 2001, Beaurepaire and Mertz, 2002, Fisher et al., 2008). This finding has sparked the development of a whole family2 of IR transmissive water immersion objectives with 16–25× magnification and NAs in the range of 0.95 and 1.1. Unfortunately, collection optical paths matching these “second generation” 2PEF objectives are challenging to design. The reason is that they have large back pupil diameters and that scattered light emerges from the objective back aperture (OBA) as a divergent cone (Oheim et al., 2001, Singh et al., 2010), calling for large-diameter optics downstream of the objective. The most common strategy to benefit from the collection power for scattered photons of these low-magnification high-NA objectives is to use proximity detectors, placed as close to the OBA as possible (Holfeld, 2007, Zinter and Levene, 2008, Durr et al., 2011). To keep the collection efficiency constant during focusing, such detectors move with the objective and have even been recommended to be an integral part of it (McMullen and Zipfel, 2010). Therefore, proximity detection schemes rarely accommodate bulky or heavy detectors, let alone multiple spectral detection channels. Lack of development of proximity detection schemes may also be due to the expense of the substantial modifications they require on commercial microscope bodies, especially when weighed against the relatively small research market that would purchase this equipment. Also, even within the two-photon user community, the benefits of efficient scattered-light collection are not always recognized.

An alternative is to use fiber-coupled detection optics: because light coupling into a large-core optical fiber requires only a large-format dichroic mirror and one or two lenses mounted close to the OBA, it permits the removal of most of the intermediate optics, fluorescence filters, and photomultiplier tubes from close to the objective. Space and weight constraints are drastically reduced (Mancuso et al., 2009). Despite their use in confocal detection (Delaney and Harris, 1995), 2PEF endoscopes and miniature head-mounted 2PEF microscopes (see Flusberg et al., 2005, Fu and Gu, 2007, Helmchen, 2002 for review), detection schemes including fiber optics have rarely been used in conventional 2PEF microscopy, probably reflecting the general belief that fiber coupling and transmission losses outweigh the benefits.

In the present paper we present two new developments based on large-core fiber-optic fluorescence detection. First, we describe a liquid light guide-coupled non-descanned detector matched to the high collection power of a 20×/NA0.95 water immersion objective that requires only minimal modification of a commercial microscope body. This fiber based detection scheme collects seven times more signal than a non-descanned detector mounted in the usual position behind the filter turret in the epi-illuminator. Thus, large-core fiber-optic detection permits, on a minimally modified commercial microscope body, a collection gain similar to that obtained with a proximity detector on a home-built set-up.

Second, we show the benefits of fiber coupled detection for spectral detection. When using a 63×/NA0.90 water immersion objective and a plastic fiber on a custom microscope (Ducros et al., 2009), we show that in addition to improving the fluorescence collection, light propagation inside the optical fiber removes the spatial sensitivity of spectral detection that can occur when photons hit the color-splitting optics with varying incidence angles, depending on their origin in the field of view.

Thus, the use of large-core optical fibers in the detection optical path can make multi-color 2PEF imaging and linear unmixing more efficient and reliable.

Section snippets

Measuring the light distribution inside the microscope

To characterize the collection efficiency of the 20×/NA0.95 objective for multiply scattered photons, we mimicked the expected spatially and angularly broad distributions of fluorescence photons at the brain surface (Beaurepaire and Mertz, 2002) with a model test sample (Fig. 1A). A large-diameter dome-shaped white light-emitting diode (LED, 6-mm ∅, radiospares), associated with a 30°-circular light-shaping diffuser (20DKIT-C3, Spectra-PhysicsNewport, Mountain View, CA) and optical paper

Microscope optical paths designed for rejecting scattered light waste most of the scattered 2PEF collected by the objective

Bulk and surface scattering determine the spatial and angular distributions of the fluorescence that exits from the brain surface and geometrical optics determines how efficiently these photons are detected (Beaurepaire and Mertz, 2002). Detection optics for biological 2PEF microscopy should be designed to collect a maximum of scattered fluorescence photons. Unlike ‘ballistic’ (non-scattered) photons that emerge from the rear of the objective as a collimated light bundle, scattered photons

Discussion

In this study, we report two new applications in which we show detection optics including a large-core fiber to be advantageous over conventional, air-coupled detection schemes for 2PEF microscopy. Robust signal gains were achieved in brain slices. The reason for the improved signal intensity is that larger acceptance angles of the detection optics can be implemented with fiber coupling. Although 63× or 40× objectives benefit already from large-θf detection optics, the realized collection gains

Author contributions

M.D. conceived and built the setup for spectral 2PEF detection. M.v.H., E.S. and M.O. conceived and built the 20×/NA0.95 setup. C.S. performed optical modeling. A.E., M.D. and M.O. performed experiments. M.D., M.O. and C.S. analyzed the data. S.C. and M.O. designed and directed research. S.C., M.D., C.S. and M.O. wrote the manuscript.

Acknowledgements

We thank Dr Jérôme Lecoq for help with LabVIEW programming, Karine Hérault, Patrice Jegouzo and Audrey Robin for excellent technical support, and our colleagues for helpful discussions and suggestions. Dr. Rainer Uhl helped with optical design. Dr. Nicole Ropert assisted in slice experiments and gave critical feedback on earlier versions of this manuscript. We acknowledge the help of JacSue Kehoe in preparing the manuscript. Brainbow-1.0 mice were a gift of Dr. Jean Livet. Supported by the

References (38)

  • C.A. Combs et al.

    Optimization of multiphoton excitation microscopy by total emission detection using a parabolic light reflector

    J Microsc

    (2007)
  • C.A. Combs et al.

    Optimizing multiphoton fluorescence microscopy light collection from living tissue by noncontact total emission detection (epiTED)

    J Microsc

    (2011)
  • P.M. Delaney et al.

    Fiberoptics in confocal microscopy

  • M. Ducros et al.

    Spectral unmixing: analysis of performance in the olfactory bulb in vivo

    PLoS ONE

    (2009)
  • N.J. Durr et al.

    Maximum imaging depth of two-photon autofluorescence microscopy in epithelial tissues

    J Biomed Opt

    (2011)
  • C.J. Engelbrecht et al.

    Enhanced fluorescence signal in nonlinear microscopy through supplementary fiber-optic light collection

    Opt Express

    (2009)
  • J.A.N. Fisher et al.

    Two-photon excitation of potentiometric probes enables optical recordings of action potentials from mammalian nerve terminals in situ

    J Neurophysiol

    (2008)
  • B.A. Flusberg et al.

    Fiber-optic fluorescence imaging

    Nat Methods

    (2005)
  • L. Fu et al.

    Fibre-optic nonlinear optical microscopy and endoscopy

    J Microsc

    (2007)
  • Cited by (0)

    1

    Present address: Dept. of Neurophysiology, Brain Research Institute, University of Zurich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.

    View full text