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Abstract
Three-dimensional (3D) reconstruction of thick samples using superresolution fluorescence microscopy remains challenging due to high level of background noise and fast photobleaching of fluorescence probes. We develop superresolution fluorescence microscopy that can reconstruct 3D structures of thick samples with both high localization accuracy and no photobleaching problem. The background noise is reduced by optically sectioning the sample using line-scan confocal microscopy, and the photobleaching problem is overcome by using the DNA-PAINT (Point Accumulation for Imaging in Nanoscale Topography). As demonstrations, we take 3D superresolution images of microtubules of a whole cell, and two-color 3D images of microtubules and mitochondria. We also present superresolution images of chemical synapse of a mouse brain section at different z-positions ranging from 0 μm to 100 μm.
Highlights
Superresolution fluorescence microscopy has made possible a variety of new discoveries previously unattainable by using conventional optical microscopes [1,2,3,4,5,6,7,8,9,10,11,12,13,14]
To characterize the effect of the asymmetric localization uncertainties in the x/y- and z-direction on the image quality, we evaluated the full width at half maximum (FWHM) of microtubule image in the blue box of Fig. 2h
It was possible by synergistically combining line-scan confocal microscopy with DNA-PAINT; while DNA-PAINT helped line-scan confocal microscopy to overcome the photobleaching problem of fluorescent probes, line-scan confocal microscopy provided the optical sectioning capability that saves DNA-PAINT method from its huge background noise
Summary
Superresolution fluorescence microscopy has made possible a variety of new discoveries previously unattainable by using conventional optical microscopes [1,2,3,4,5,6,7,8,9,10,11,12,13,14]. Current application of superresolution fluorescence microscopy to 3D reconstruction of specimens, is limited to thin samples. If we can apply superresolution fluorescence microscopy to reconstruct 3D structures of thick tissue samples, it will revolutionize biological studies including studies of organism developments and neural connectomics. Two main barriers need to be overcome for superresolution fluorescence microscopy to be successfully used for optical 3D reconstruction of thick biological samples.
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