Reflective imaging improves spatiotemporal resolution and collection efficiency in light sheet microscopy

Light-sheet fluorescence microscopy (LSFM) enables high-speed, high-resolution, and gentle imaging of live 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 four complementary views in 250 ms, doubling speed and improving information content relative to symmetric dual-view LSFM. We also report a modified deconvolution algorithm that removes 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 single-view LSFM, permitting simultaneous acquisition of two high-resolution views otherwise difficult to obtain due to steric constraints at high numerical aperture. We demonstrate the broad applicability of our method in a variety of samples, studying mitochondrial, membrane, Golgi, and microtubule dynamics in cells and calcium activity in nematode embryos.

File Name: Supplementary Movie 3 Description: Time-lapse reflective imaging of a 3-fold nematode embryo expressing GCaMP3 under the myo-3 promoter acquired with 0.8/0.8 NA diSPIM, without rolling shutter slit detection. We achieved a continuous volumetric imaging rate of 2.85 Hz (250 ms for quad view acquisition and 100 ms settling time for the piezo stage to translate back to its initial position). Maximum intensity XY and ZY projections of deconvolved reconstructions (with respect to the coordinate system of the objective) are shown, indicating rapid calcium flux within the embryo muscles. Time is indicated in seconds. See also Fig. 2.
File Name: Supplementary Movie 4 Description: Time-lapse reflective imaging of a 3-fold nematode embryo expressing GCaMP3 under the myo-3 promoter acquired with 0.8/0.8 NA diSPIM with rolling shutter slit detection. The embryo was imaged every 600 ms, for 600 volumes. Maximum intensity XY and ZY projections of deconvolved reconstructions (with respect to the coordinate system of the objective) are shown. Time is indicated in seconds. See also Fig. 2.
File Name: Supplementary Movie 5 Description: Reflective light sheet volumetric time lapse imaging showing TurboGFP-Lck (cyan, staining the plasma membrane, endocytic machinery, and Golgi compartment) and Tom20-mApple (magenta, staining the outer mitochondrial membrane) in a U2OS cell, acquired with 0.8/0.8 NA diSPIM. XY views at three depths (0, 1.1, and 3. 7 µm depth, measured and viewed from the coverslip surface) are shown. The cell was imaged every 5 seconds over 25 minutes (400 ms to simultaneously acquire four dual-color views, and 5 seconds inter-volume pause). Time is indicated as minutes : seconds. See also File Name: Supplementary Movie 11 Description: Asymmetric reflective diSPIM imaging showing GFP-labeled Golgi in a U2OS cell, revealing highly dynamic, ribbon-like Golgi stacks juxtaposed around the nucleus, as well as rapidly moving Golgi vesicles. Maximum intensity projections (XY and ZY views, from the perspective of reflective coverslips) are shown. The cell was imaged every 15 seconds for 45 minutes (i.e., 180 volumes, each volume containing two 1.1 NA views was acquired within 1 second). Time is indicated as minutes: seconds.
File Name: Supplementary Movie 12 Description: Asymmetric reflective diSPIM imaging showing a 3-fold nematode embryo expressing GCaMP3 from the nmr-1 promoter, highlighting calcium transient during backwards movement. Maximum intensity projections (XY and ZY views from the perspective of reflective coverslips) are shown. The embryo was imaged every 640 ms for 32 seconds (i.e., 50 volumes, 90 planes/volume, each volume containing two 1.1 NA views acquired within 540 seconds). Time is indicated in seconds. See also Fig. 5d-i.
File Name: Supplementary Software 1 Description: The software is compressed as a zip file. It includes four main MATLAB scripts for the deconvolution of conventional diSPIM imaging on glass coverslips (Fig. 1e, Fig. 4d-f, Supplementary Fig. 6, Supplementary Movie 7), symmetric diSPIM on reflective coverslips ( Fig. 1f-g, Fig. 2, Fig. 3, Supplementary Fig. 2, Supplementary Movies 3-6), dual-view asymmetric diSPIM on reflective coverslips (Fig. 4d, Fig. 4g, Fig. 5, Supplementary Movies 8-12), and quadruple-view asymmetric diSPIM on reflective coverslips (Fig. 4g), respectively. It also includes four accessory MATLAB scripts, two for reading and writing TIFF stacks, one for performing 3D convolution in the Fourier domain, and one for computing the excitation light sheet pattern. Finally, it includes a text file that explains how to run the code.