Abstract
Light-sheet fluorescence microscopy (LSFM), also termed single plane illumination microscopy (SPIM), enables live cell fluorescence imaging with optical sectioning capabilities superior to confocal microscopy and without any out-of-focus exposure of the specimen. However, the need of two objective lenses, one for light-sheet illumination and one for imaging, imposes geometrical constraints that require LSFM setups to be adapted to the specific needs of different types of specimen in order to obtain optimal imaging conditions. Here we demonstrate the use of an oblique light-sheet configuration adapted to provide the highest possible Gaussian beam enabled resolution in LSFM. The oblique light-sheet configuration furthermore enables LSFM imaging at the surface of a cover slip, without the need of specific sample mounting. In addition, the system is compatible with simultaneous high NA wide-field epi-fluorescence imaging of the specimen contained in a glass-bottom cell culture dish. This prevents cumbersome sample mounting and enables rapid screening of large areas of the specimen followed by high-resolution LSFM imaging of selected cells. We demonstrate the application of this microscope for in toto imaging of endocytosis in yeast, showing for the first time imaging of all endocytic events of a given cell over a period of >5 minutes with sub-second resolution.
Highlights
Light-sheet fluorescence microscopy (LSFM), termed single plane illumination microscopy (SPIM), enables live cell fluorescence imaging with optical sectioning capabilities superior to confocal microscopy and without any out-of-focus exposure of the specimen
A non-orthogonal arrangement allows for free adjustment of light-sheet angle and NA within the bounds of the free angular range of a given detection objective (Fig. 1), with the sum of the opening angles 2(αNAexc. +αNAdet.) approaching π. πSPIM uses an inverted arrangement of an oil immersion objective lens, which serves for wide-field imaging and for oblique light-sheet generation simultaneously
The resolution in light-sheet fluorescence microscopy (LSFM) is characterized by its effective point-spread function (PSF), which results from the overlap of the light-sheet used for illumination and the PSF of the detection objective lens
Summary
Light-sheet fluorescence microscopy (LSFM), termed single plane illumination microscopy (SPIM), enables live cell fluorescence imaging with optical sectioning capabilities superior to confocal microscopy and without any out-of-focus exposure of the specimen. With the use of high-NA (≥1 .4) oil immersion objective lenses for light-sheet generation, the light-sheet NA can be freely adjusted to fill all of the angular range left available by the detection objective Such an arrangement allows generous access to the specimen and importantly, it permits it to reside on the surface of the cover slip (e.g. adherent cells). This is a prerequisite for simultaneous high-NA wide-field imaging through the illumination objective and can be combined with a wide-field set-up for convenient selection of specific areas in the full FOV of the specimen, such as individual cells, for their subsequent analysis using light-sheet imaging. This is useful when light-sheet microscopy is employed to study a rare subpopulation among the cells present in the sample
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