Abstract
Three-dimensional fluorescence-based imaging of living cells and organisms requires the sample to be exposed to substantial excitation illumination energy, typically causing phototoxicity and photobleaching. Light sheet fluorescence microscopy dramatically reduces phototoxicity, yet most implementations are limited to objective lenses with low numerical aperture and particular sample geometries that are built for specific biological systems. To overcome these limitations, we developed a single-objective light sheet fluorescence system for biological imaging based on axial plane optical microscopy and digital confocal slit detection, using either Bessel or Gaussian beam shapes. Compared to spinning disk confocal microscopy, this system displays similar optical resolution, but a significantly reduced photobleaching at the same signal level. This single-objective light sheet technique is built as an add-on module for standard research microscopes and the technique is compatible with high-numerical aperture oil immersion objectives and standard samples mounted on coverslips. We demonstrate the performance of this technique by imaging three-dimensional dynamic processes, including bacterial biofilm dispersal, the response of biofilms to osmotic shocks, and macrophage phagocytosis of bacterial cells.
Highlights
Live-cell optical microscopy is a powerful tool for characterizing the dynamics of life in three dimensions (3D)
We implemented a digital-scanning confocal light sheet imaging technique that is based on axial plane optical microscopy (APOM), which combines the benefits of selective plane illumination, confocal detection, high-numerical aperture (NA) objectives, and standard sample geometries based on coverslip mounting
The digital confocal light-sheet axial plane optical microscopy (DC-APOM) technique is compatible with 96-well plates, microfluidic chambers, and standard sample geometries that can be used on standard confocal microscopes
Summary
Live-cell optical microscopy is a powerful tool for characterizing the dynamics of life in three dimensions (3D). Living samples are typically photosensitive and traditional 3D imaging techniques, such as confocal microscopy or two-photon microscopy, cause significant phototoxicity. This phototoxicity can interfere with biological systems, when samples are imaged with high temporal resolution for extended periods. Light-sheet fluorescence microscopy (LSFM) or selective plane illumination microscopy (SPIM) have re-emerged as important imaging modalities for 3D live-cell imaging, due to their strongly reduced phototoxicity compared with other 3D imaging techniques [1,2,3,4,5]. The volume of the sample that is illuminated by excitation light in confocal microscopy or two-photon microscopy is significantly larger than the volume that is imaged at a given time, resulting in stronger phototoxicity and photobleaching
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