Real-time multi-angle projection imaging of biological dynamics

Abstract

We introduce a cost-effective and easily implementable scan unit that converts any camera-based microscope with optical sectioning capability into a multi-angle projection imaging system. Projection imaging reduces data overhead and accelerates imaging by a factor of >100, while also allowing users to readily view biological phenomena of interest from multiple perspectives on the fly. By rapidly interrogating the sample from just two perspectives, our method also enables real-time stereoscopic imaging and three-dimensional particle localization. We demonstrate projection imaging with spinning disk confocal, lattice light-sheet, multidirectional illumination light-sheet and oblique plane microscopes on specimens that range from organelles in single cells to the vasculature of a zebrafish embryo. Furthermore, we leverage our projection method to rapidly image cancer cell morphodynamics and calcium signaling in cultured neurons at rates up to 119 Hz as well as to simultaneously image orthogonal views of a beating embryonic zebrafish heart.

Figure 1 -. Principle of multi angle projection imaging.

A Illustration of the shear-warp transform. Top row: simulated volume undergoing shearing. Middle row: simulated volume undergoing rotation. Bottom: shadows (projections) cast by the volumes are identical up to a scaling factor. B In a conventional fluorescence microscope, the focal plane is scanned relative to the sample along the optical axis. C The images acquired in this way (green tiles) are stacked in the z-dimension one after the other to form a “3D stack”. C Experimental data stack acquired with LLSM and its projection. E For projection imaging under different viewing angles, a lateral shearing unit consisting of two galvanometric mirrors is added in front of the camera. When the sample is scanned (blue arrow), these two mirrors are rotated in synchrony, causing the image to be displaced laterally on the camera (black double headed arrow). F The images acquired this way (green tiles) are stacked in the z (blue arrow, labeled “stacking”) and laterally shifted in the x-direction (black arrow, labeled “shear”). G Same experimental data as in D, but after applying shearing. D and G A projection can be computed by numerically summing all the tiles in the 3D stack together or by scanning the sample one or multiple times during one camera exposure. H Projection views of an MV3 cell under different shearing parameters (viewing angle measured to the normal of the coverslip), acquired with LLSM-Pro. Only one image was acquired for each view. Scale bars: 10 microns.

Figure 2 –. Projection imaging applied to various microscopes.

A Numerical projection of a conventional z-stack (334 planes) of MV3 cells labeled with AKT-PH-GFP, acquired with conventional LLSM. B Projection view of an MV3 cells expressing AKTPH-GFP, acquired with LLSM-Pro. This view was acquired on a single camera frame. C Vasculature in the hindbrain of a Zebrafish embryo, viewed under a 45-degree angle to the optical axis, as imaged with mSPIM-Pro. The veins/endothelial tissue of the zebrafish was labeled with GFP (Tg(krdl:GFP)). D Spheroid formed by cancer cells (A-375) labeled with GFP-Tractin under two viewing angles (45 and 0 degrees) as imaged by mSPIM-Pro. E Mouse colon crypt cells labeled with cytosolically with TdTomato and embedded in collagen as imaged by SDCM-Pro. F single slice view of cultured Neurons, acquired with OPM. Only a narrow field of view is visible at any time G Projection view of cultured neurons at the same location as in G, acquired with OPM-Pro. Scale bars: A,B,E,F 10 microns. C,D 50 microns

Figure 3 -. Dynamic projection imaging.

A MV3 cell labeled with AKT-PH-GFP as imaged with LLSM-Pro. B&C Zoomed in version of the regions within the blue and green boxes in A. D&E Kymographs along the red dotted lines shown in B&C. The orange dotted lines indicate different growth phases in two blebs. F Projection of cultured cortical neurons expressing the calcium indicator GCaMP6f as imaged by OPM-Pro. G Signal trace from the oval region marked in F displayed as the change in fluorescence intensity (ΔF) relative to its baseline value (F). Scale bars: A-C 10 microns; F 20 microns.

Figure 4 –. Simultaneous Multi Angle Viewing.

A Schematic illustration of two viewing directions of a cell on a coverslip. B Anaglyph for 3D viewing of a MV3 cancer cells expressing AKT-PH GFP obtained with LLSM-Pro. 3D red cyan glasses are recommended to view this image correctly. The image was created by two different views (15° and −15° to the normal of the coverslip) of the cell at a frame rate of 1Hz. C 3D localization from two views of genetically encoded monomeric nanoparticles in a fixed MV3 cell. Magenta shows a rendering of the location in 3D, and maximum intensity projections are shown on the side in black. Small inset shows the two projections (green and magenta) obtained with OPM-Pro. The difference in viewing angle between the two views amounts to 18 degrees. D Schematic illustration of a dual camera setup that allows simultaneous acquisition of projections under two different viewing angles. E Orthogonal viewing directions under which an embryonic zebrafish heart was imaged. F Three frames of a movie of orthogonal projections of a beating zebrafish heart imaged at a framerate of 10 Hz, obtained with LLSM-Pro. The veins/endothelial tissue of the zebrafish was labeled with GFP (Tg(krdl:GFP)). View 1 is −60 degrees and view 2 is 30 degrees relative to the nominal focal plane of the detection objective. Scale bars: 10 microns in B-C and 20 microns in F.