Abstract
The development of imaging techniques beyond the diffraction limit has paved the way for detailed studies of nanostructures and molecular mechanisms in biological systems. Imaging thicker samples, such as mammalian cells and tissue, in all three dimensions, is challenging due to increased background and volumes to image. Light sheet illumination is a method that allows for selective irradiation of the image plane, and its inherent optical sectioning capability allows for imaging of biological samples with reduced background, photobleaching, and photodamage. In this review, we discuss the advantage of combining single-molecule imaging with light sheet illumination. We begin by describing the principles of single-molecule localization microscopy and of light sheet illumination. Finally, we present examples of designs that successfully have married single-molecule super-resolution imaging with light sheet illumination for improved precision in mammalian cells.
Highlights
Fluorescence microscopy in combination with specific labeling of biomolecules has been a standard tool for elucidating molecular mechanisms in biological systems for many decades
This field was advanced further by the development of far-field super-resolution fluorescence techniques, which allow for imaging beyond the diffraction limit by using e.g. stimulated emission depletion microscopy (STED) [4,5], structured illumination microscopy (SIM) [6,7], or single-molecule active-control techniques, such as photoactivated localization microscopy ((f)PALM) [8,9] and stochastic optical reconstruction microscopy (STORM) [10]
A third issue with excessive illumination is increased risk of collateral photodamage when imaging sensitive live samples. These issues can all be mitigated by combining single-molecule super-resolution imaging with light sheet illumination [19], where the sample is optically sectioned by a sheet of light illuminating the image plane of the detection optics
Summary
Fluorescence microscopy in combination with specific labeling of biomolecules has been a standard tool for elucidating molecular mechanisms in biological systems for many decades. When imaging thick samples, such as mammalian cells, tissue, and polymers and gels, background fluorescence arising from out-of-focus emitters reduces the ability to detect and localize individual molecules precisely. This is especially true when imaging samples in all three dimensions (3D). A third issue with excessive illumination is increased risk of collateral photodamage (e.g. due to formation of reactive oxygen species) when imaging sensitive live samples These issues can all be mitigated by combining single-molecule super-resolution imaging with light sheet illumination [19], where the sample is optically sectioned by a sheet of light illuminating the image plane of the detection optics. We will give examples of methods that successfully have married single-molecule super-resolution imaging with light sheet illumination for improved precision in mammalian cells
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