Single-objective high-resolution confocal light sheet fluorescence microscopy for standard biological sample geometries
Stoyan Yordanov, Konstantin Neuhaus, Raimo Hartmann, Francisco Díaz-Pascual, Lucia Vidakovic, Praveen K. Singh, Knut Drescher
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.