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The DIVER Microscope for Imaging in Scattering Media

1
Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California Irvine, Irvine, CA 92697, USA
2
Departamento de Fisiopatología, Hospital de Clínicas, Facultad de Medicina, Universidad de la República-Uruguay, Montevideo 11400, Uruguay
*
Author to whom correspondence should be addressed.
Methods Protoc. 2019, 2(2), 53; https://doi.org/10.3390/mps2020053
Received: 11 May 2019 / Revised: 6 June 2019 / Accepted: 19 June 2019 / Published: 21 June 2019
(This article belongs to the Special Issue Technical Advances in Light Microscopy)
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Abstract

We describe an advanced DIVER (Deep Imaging Via Emission Recovery) detection system for two-photon fluorescence microscopy that allows imaging in multiple scattering media, including biological tissues, up to a depth of a few mm with micron resolution. This detection system is more sensitive to low level light signals than conventional epi-detection used in two-photon fluorescence microscopes. The DIVER detector efficiently collects scattered emission photons from a wide area of turbid samples at almost any entrance angle in a 2π spherical angle. Using an epi-detection scheme only photons coming from a relatively small area of a sample and at narrow acceptance angle can be detected. The transmission geometry of the DIVER imaging system makes it exceptionally suitable for Second and Third Harmonic Generation (SHG, THG) signal detection. It also has in-depth fluorescence lifetime imaging (FLIM) capability. Using special optical filters with sin-cos spectral response, hyperspectral analysis of images acquired in-depth in scattering media can be performed. The system was successfully employed in imaging of various biological tissues. The DIVER detector can be plugged into a standard microscope stage and used as an external detector with upright commercial two-photon microscopes. View Full-Text
Keywords: non-linear microscopy; hyperspectral imaging; FLIM; scattering media; fluorescence non-linear microscopy; hyperspectral imaging; FLIM; scattering media; fluorescence
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Dvornikov, A.; Malacrida, L.; Gratton, E. The DIVER Microscope for Imaging in Scattering Media. Methods Protoc. 2019, 2, 53.

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