Abstract
.We demonstrate improved optical sectioning in light sheet fluorescence microscopy using tunable structured illumination (SI) frequencies to optimize image quality in scattering specimens. The SI patterns are generated coherently using a one-dimensional spatial light modulator for maximum pattern contrast, and the pattern spatial frequency is adjustable up to half the incoherent cutoff frequency of our detection objective. At this frequency, we demonstrate background reductions of 2 orders of magnitude.
Highlights
Light sheet fluorescence microscopy (LSFM) has transformed imaging in the life sciences with its ability to rapidly acquire optical sections in thick specimens.[1]
The suspension is composed of water, 5% Intralipid, and 1% agarose, which results in a matrix with a scattering coefficient of roughly 100 cm−1, similar to many human tissues.[11]
To limit the effect of photobleaching on repeated scanning of the same volume, the power is kept low and frame rate limited to 75 frames per second
Summary
Light sheet fluorescence microscopy (LSFM) has transformed imaging in the life sciences with its ability to rapidly acquire optical sections in thick specimens.[1] LSFM uses lower peak intensities than other optical sectioning techniques, leading to less photobleaching. The high speed and favorable photobleaching properties of LSFM make it a powerful volumetric reconstruction tool. LSFM has no inherent background rejection mechanism. When imaging in scattering samples, the light sheet broadens as it travels, leading to large out-of-focus components. Samples are often optically cleared to circumvent this problem, but high-quality hydrogel-based clearing techniques, such as CLARITY, typically require days to weeks for processing.[2]
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