Abstract
Fluorescence microscopy is a powerful approach for studying subcellular dynamics at high spatiotemporal resolution; however, conventional fluorescence microscopy techniques are light-intensive and introduce unnecessary photodamage. Light-sheet fluorescence microscopy (LSFM) mitigates these problems by selectively illuminating the focal plane of the detection objective by using orthogonal excitation. Orthogonal excitation requires geometries that physically limit the detection objective numerical aperture (NA), thereby limiting both light-gathering efficiency (brightness) and native spatial resolution. We present a novel live-cell LSFM method, lateral interference tilted excitation (LITE), in which a tilted light sheet illuminates the detection objective focal plane without a sterically limiting illumination scheme. LITE is thus compatible with any detection objective, including oil immersion, without an upper NA limit. LITE combines the low photodamage of LSFM with high resolution, high brightness, and coverslip-based objectives. We demonstrate the utility of LITE for imaging animal, fungal, and plant model organisms over many hours at high spatiotemporal resolution.
Highlights
To properly visualize and measure cellular and subcellular dynamics, cell biologists demand imaging at high spatial and temporal resolution
lateral interference tilted excitation (LITE) illuminates a thin slice of fluorescent samples The feature shared by all SPIM/Light-sheet fluorescence microscopy (LSFM) technologies is the spatial restriction of the illumination light to a volume on the order of magnitude of the detection objective’s focal plane, so that fluorophores outside of the focal plane do not experience unnecessary illumination
LSFM has been used to reduce photodamage to live fluorescent samples by reducing the illumination to only the focal volume of the detection objective (Santi, 2011), but its geometry has prevented the use of high-numerical aperture (NA) objective lenses
Summary
To properly visualize and measure cellular and subcellular dynamics, cell biologists demand imaging at high spatial and temporal resolution. Conventional fluorescence microscope modalities require high-intensity light to illuminate the sample through the objective lens, exciting all fluorophores in the path of the collimated excitation light. The fluorophores emit light that is collected by the objective lens and transmitted to the detector. A disadvantage of the traditional “epi-illumination” geometry is that light is emitted from fluorophores outside the focal plane and contributes to the image, which confounds the focal information. Confocal microscopy mitigated this problem by selectively collecting light from the focal plane through the use of conjugate pinholes (Stelzer et al, 1995). The most common method for reducing both out-of-focus excitation and emission, total internal reflection fluorescence microscopy, can only illuminate regions of the cell within ∼200 nm of the coverslip surface (Axelrod, 1981)
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