Concept of projections with parameter selection (props).

a Schematic overview of the imaging geometry in oblique plane microscopy (OPM) and lattice light-sheet microscopy (LLSM). The sample is rapidly scanned with a light sheet (blue) and corresponding focal plane, which is termed a “focal sweep”. If the focal sweep is completed during one camera exposure, an optical projection is formed. b Translating the image on the camera (red arrow labeled “shear”) during a focal sweep results in projections under different viewing angles. On the right, a top-down projection along the z-axis is shown. c When a rolling shutter (red boxes) is synchronized to the shear translation along the camera, a projection of a sub-volume (red shaded area in a) is formed. d, e Schematic illustration of varying the projection depth with the rolling shutter width. The red-shaded volumes illustrate the regions of the sample that are projected below, illustrated as a top-down view. f Illustration of shifting the projection volume axially by introducing a time delay between the rolling shutter and the focal sweep and shearing. g A nonlinear scanning waveform results in an axial change within the projection volume. h LLSMprops imaging of A375 cells labeled with F-tractin-EGFP with varying projection depth, top row: top-down view, bottom row: corresponding 45° viewing angle. The imaging experiment was repeated independently 3 times with similar results. i, j Mesoscopic OPMprops imaging of zebrafish vasculature, labeled with Tg(kdrl:EGFP), over a projection depth of 4 and 362 microns, respectively. The imaging experiment was repeated independently 4 times with similar results. Scale Bar: h: 10 microns, j: 500 microns.

Variation of projection parameters on different biological samples.

a Projection of a Drosophila embryo labeled with UAS-actin5C-RFP using OPMpro. b OPMprops projection of the same embryo. c Projection of the gill vasculature labeled with the vascular marker Tg(kdrl:Hsa.HRAS-mCherry) in a zebrafish larva using OPMpro. d Three sequentially acquired projections using OPMprops. The projections divide the volume in c into sub-compartments, which are color-coded in magenta, green, and cyan, respectively. Both the Drosophila and zebrafish imaging were repeated independently 5 times with similar results. e A single osteosarcoma (U-2 OS) cell, as imaged with oblique plane structured illumination microscopy (OPSIM) using props. Green are mitochondria in a projection layer adjacent to the coverslip, magenta are mitochondria in a projection layer 4 microns above the coverslip. The two projections were acquired sequentially. The imaging experiment was repeated independently 3 times with similar results. f Schematic representation of imaging an x–y layer (orange) near the bottom of a Drosophila embryo (gray) that is mapped in a top-down view (dotted arrow). g Schematic representation of a curved surface (blue) that is mapped into a planar projection (dotted arrow). h XY projection of a Drosophila embryo labeled with myosin-FRB-GFP. i Curved projection of the same embryo. Red arrows point to selected features visible in both (h) and (i). The imaging experiment was repeated independently 4 times with similar results. Scale Bars: a–c: 50 microns, e: 10 microns, i: 50 microns.

Selective projection imaging of calcium dynamics.

a–d Calcium dynamics in a Drosophila embryo labeled with jGCaMP7s-CAAX imaged at a rate of 50 Hz using OPMprops. b Zoomed-in views of the area surrounding the dashed line in (a). c Kymograph along the dotted line in (a). d Calcium signal analysis (dF/F) from the box in (c). e Projection image of another jGCaMP7s-CAAX labeled embryo using OPMpro. f Same embryo as in (e), but imaged with OPMprops at 20 Hz framerate. g Kymograph along the dotted line in (e). h Kymograph along the dotted line in (f). i–n OPMprops imaging at two depths within a zebrafish larva labeled with Tg(elavl3:soma-GCaMP7f). i Overlay of the two projections. j, k Individual projections. l Schematic representation of the projection imaging. The two sub-volumes (“slabs”) are separated by 40 microns. m–n Color-coded timeseries corresponding to j, k where the static background was subtracted. Both the Drosophila embryo and zebrafish larva imaging were repeated independently 3 times with similar results. Source data are provided as a Source Data file.