High-speed 3D light-sheet microscopy

Light-sheet fluorescence microscopy (LSFM) provides low out-of-plane photobleaching and phototoxicity and is therefore well suited to high-speed optically-sectioned imaging of biological specimens in 2D or 3D. Conventional LSFM configuration employs two microscope objective lenses orientated at 90° to one another; the first is used to generate an illumination light sheet and the second is used to collect fluorescence from the illuminated plane. However, in order to be able to scan the detection plane though the sample rapidly, some form of optical remote-refocussing is required.

My lab has explored a number of approaches for video-rate 3D LSFM. One approach is the use of adaptive-optics by employing a deformable mirror (DM). This allows the collected fluorescence to be refocussed very efficiently, but is technically challenging to implement. An alternative approach is the refocussing approach of Botcherby et al. in the detection path of LSFM. This approach allows a larger field of view compared to the DM-based approach, but the emitted fluorescence is only partly collected due to a polarisation beam splitter in the emission path. A third approach is oblique plane microscopy (OPM), which uses a single high numerical aperture microscope objective to provide both fluorescence excitation and detection whilst maintaining the advantages of LSFM, enabling it to provide high-speed 3D imaging for a range of applications on a conventional fluorescence microscope frame. This talk will review these different approaches and give examples of their application in biology.