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