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The DIVER Microscope for Imaging in Scattering Media.

Alexander Dvornikov1, Leonel Malacrida2,3, Enrico Gratton4

  • 1Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California Irvine, Irvine, CA 92697, USA. advornik@uci.edu.

Methods and Protocols
|June 26, 2019
PubMed
Summary
This summary is machine-generated.

We developed the Deep Imaging Via Emission Recovery (DIVER) system for advanced two-photon microscopy. This system enables high-resolution imaging deep within scattering biological tissues, improving sensitivity for low light signals.

Keywords:
FLIMfluorescencehyperspectral imagingnon-linear microscopyscattering media

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Area of Science:

  • Biomedical Optics
  • Microscopy Technology
  • Photonics

Background:

  • Two-photon fluorescence microscopy is limited in imaging depth due to scattering in biological tissues.
  • Conventional epi-detection methods in two-photon microscopy have limited sensitivity and collection angles for scattered photons.
  • Imaging deep within scattering media requires advanced detection systems capable of collecting weak, scattered signals.

Purpose of the Study:

  • To introduce and characterize the Deep Imaging Via Emission Recovery (DIVER) detection system.
  • To demonstrate DIVER's capability for high-resolution imaging in multiple scattering media, including biological tissues.
  • To highlight DIVER's advantages over conventional epi-detection for two-photon fluorescence microscopy.

Main Methods:

  • The DIVER system utilizes a wide-area photon collection geometry in a transmission setup.
  • It employs specialized optical filters for hyperspectral analysis.
  • The system is designed for integration with commercial upright two-photon microscopes.

Main Results:

  • DIVER enables imaging up to a depth of a few millimeters with micron resolution in scattering media.
  • The system exhibits higher sensitivity to low light signals compared to conventional epi-detection.
  • DIVER efficiently collects scattered emission photons from a wide sample area at various angles.
  • Successful imaging of various biological tissues was achieved.
  • The system supports Second and Third Harmonic Generation (SHG, THG) signal detection and in-depth fluorescence lifetime imaging (FLIM).

Conclusions:

  • The DIVER detection system significantly enhances the capabilities of two-photon fluorescence microscopy for deep tissue imaging.
  • Its superior sensitivity and photon collection efficiency allow for unprecedented imaging in turbid biological samples.
  • DIVER offers a versatile platform for advanced imaging modalities, including SHG, THG, and FLIM, in scattering media.