Simultaneous multi-color optical sectioning fluorescence microscopy with wavelength-coded volume holographic gratings

Opt Express. 2020 Dec 7;28(25):37177-37187. doi: 10.1364/OE.409179.

Abstract

Optical sectioning fluorescence microscopy provides high contrast images of volumetric samples and has been widely used for many biological applications. However, simultaneously acquiring multi-color fluorescence images require additional optical elements and devices, which are bulky, wavelength specific, and not cost-effective. In this paper, wavelength-coded volume holographic gratings (WC-VHGs) based optical sectioning fluorescence microscopy is proposed to simultaneously offer multi-color fluorescence images with fine out-of-focus background rejection. Due to wavelength degeneracy, multiplexed WC-VHGs are capable of acquiring multi-wavelength fluorescence images in a single shot, and displaying the laterally separated multi-wavelength images onto CCD. In our system optical sectioning capability is achieved through speckle illumination and HiLo imaging method. To demonstrate imaging characteristics of our system, dual-wavelength fluorescence images of both standard fluorescent microspheres and ex vivo mT/mG mice cardiac tissue are presented. Current results may find important applications in hyperspectral imaging for biomedical research.

MeSH terms

  • Animals
  • Fluorescence
  • Heart / diagnostic imaging*
  • Holography / methods*
  • Image Processing, Computer-Assisted
  • Lighting / instrumentation
  • Mice
  • Microscopy, Fluorescence / methods*
  • Microspheres
  • Optical Imaging / methods
  • Optical Phenomena