Photoswitching-Enabled Contrast Enhancement in Light Sheet Fluorescence Microscopy Articles uri icon

authors

  • VETTENBURG, TOM
  • CORRAL GARCIA-HERAS, ANGELICA
  • RODRIGUEZ PULIDO, ALBERTO
  • FLORS, CRISTINA
  • RIPOLL LORENZO, JORGE

publication date

  • March 2017

start page

  • 424

end page

  • 428

issue

  • 3

volume

  • 4

international standard serial number (ISSN)

  • 2330-4022

abstract

  • Light sheet fluorescence microscopy enables high-resolution imaging of thick biological samples. By restricting the fluorescence excitation to a single plane, rapid wide-field image acquisition is possible with minimal sample exposure. Although light sheet microscopy is able to resolve subcellular features at depth in model organisms, elevated levels of endogenous autofluorescence often preclude acceptable contrast and may obscure features of interest in general samples. Here we demonstrate how photoswitchable fluorophores can be exploited to boost contrast in light sheet microscopy. The novel detection method enables high specificity while maintaining the optical sectioning capability of the light sheet microscope. Our experiments reveal structures hidden well below the ambient fluorescent background level by enhancing the contrast by 2 orders of magnitude.

keywords

  • selective plane illumination microscopy; photoswitching; optical lock-in detection; autofluorescence; intensity evolution; plane illumination microscopy; lock-in detection; imaging microscopy; diffraction limit; fluorophores; specimens; proteins