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bioRxiv · 10.1101/154807

Reflective imaging improves resolution, speed, andcollection efficiency in light sheet microscopy

Wu, Y.·Kumar, A.·Smith, C.·Ardiel, E.·Chandris, P.·Christensen, R.·Rey-Suarez, I.·Guo, M.·Vishwasrao, H.·Chen, J.·Tang, J.·Upadhyaya, A.·La Riviere, P.·Shroff, H.
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Abstract

Light-sheet fluorescence microscopy (LSFM) enables high-speed, high-resolution, gentle imaging of live biological specimens over extended periods. Here we describe a technique that improves the spatiotemporal resolution and collection efficiency of LSFM without modifying the underlying microscope. By imaging samples on reflective coverslips, we enable simultaneous collection of multiple views, obtaining 4 complementary views in 250 ms, half the period it would otherwise take to collect only two views in symmetric dual-view selective plane illumination microscopy (diSPIM). We also report a modified deconvolution algorithm that removes the associated epifluorescence contamination and fuses all views for resolution recovery. Furthermore, we enhance spatial resolution (to < 300 nm in all three dimensions) by applying our method to a new asymmetric diSPIM, permitting simultaneous acquisition of two high-resolution views otherwise difficult to obtain due to steric constraints at high numerical aperture (NA). We demonstrate the broad applicability of our method in a variety of samples of moderate (< 50 m) thickness, studying mitochondrial, membrane, Golgi, and microtubule dynamics in single cells and calcium activity in nematode embryos.

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Wu, Y., Kumar, A., Smith, C., Ardiel, E., Chandris, P., Christensen, R., Rey-Suarez, I., Guo, M., Vishwasrao, H., Chen, J., Tang, J., Upadhyaya, A., La Riviere, P., Shroff, H.. 2017-06-23. Reflective imaging improves resolution, speed, andcollection efficiency in light sheet microscopy. https://doi.org/10.1101/154807

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