Dual-view plane illumination microscopy for rapid and spatially isotropic imaging

Abstract

We describe the construction and use of a compact dual-view inverted selective plane illumination microscope (diSPIM) for time-lapse volumetric (4D) imaging of living samples at subcellular resolution. Our protocol enables a biologist with some prior microscopy experience to assemble a diSPIM from commercially available parts, to align optics and test system performance, to prepare samples, and to control hardware and data processing with our software. Unlike existing light sheet microscopy protocols, our method does not require the sample to be embedded in agarose; instead, samples are prepared conventionally on glass coverslips. Tissue culture cells and Caenorhabditis elegans embryos are used as examples in this protocol; successful implementation of the protocol results in isotropic resolution and acquisition speeds up to several volumes per s on these samples. Assembling and verifying diSPIM performance takes ∼6 d, sample preparation and data acquisition take up to 5 d and postprocessing takes 3-8 h, depending on the size of the data.

Figure 1

DiSPIM with major subassemblies. (a) Perspective view of assembled diSPIM, with microscope frame, inverted microscope components, cameras and major diSPIM subassemblies indicated. ‘DiSPIM module’ refers to scanners, dichroic filter cubes, tube lenses, objectives, piezos and associated optomechanics that move as a single unit on an LS-50 translation stage. Refer to the MATERIALS section for further description of component parts. (b) Photograph to accompany a. (c) Higher-magnification view of excitation and detection subassemblies in the diSPIM module. Excitation (blue) and emission (red) along a single arm of the module are highlighted. (d) Detailed view of the excitation scanner, showing fiber input, mirrors and relay lenses. (e) Higher-magnification view of objectives, piezos and holders/adjusters. (f) Photograph to accompany e. See also Supplementary Note 1, SF2 and SF3.

Figure 2

Verifying system performance. (a) Measuring the system PSF across the FOV by collecting a Z stack on a fluorescent bead. Light sheet and the collection objective move in sync. (b) XY maximum intensity projections of beads, showing raw data from view A (left) and view B (right). Scale bar, 5 μm. (c) Higher-magnification view of beads within dashed rectangular region in b, highlighting the improvement in lateral and axial resolution after joint deconvolution procedure. Scale bar, 3 μm. See also Supplementary Table 4 with lateral and axial resolution from all beads marked with red circles in b. (d) Measuring the light sheet thickness. The collection objective remains focused on the fluorescent bead, integrating the fluorescence as the light sheet is scanned through the bead. (e) Light sheet thicknesses in arm A (blue diamonds) and arm B (red squares). Apparent light sheet thicknesses at this location are 2.1 μm (SPIM A) and 1.8 μm (SPIM B). Data were derived from the bead marked with the blue squares in b. See also Supplementary Note 1, SF13, showing the light sheet thickness measured at different locations across the FOV. Note that schematics a and d are not to scale; dimensions have been altered to improve image clarity. See also Supplementary Data 2–5.

Figure 3

Imaging labeled histones in a C. elegans embryo with diSPIM. Selected maximum intensity projections from ~14-h imaging experiment, showing nuclear histones in a living C. elegans embryo. Data indicate the isotropic resolution expected from a properly functioning diSPIM system, as well as the ability to image samples over many time points with negligible bleaching and photodamage. Time is indicated as hours post fertilization (h.p.f.) in h:min:s format. See also Supplementary Video 1, Supplementary Data 1 and 7.

Figure 4

Imaging GFP-tagged Harvey rat sarcoma viral oncogene homolog (HRAS) in transformed human lung fibroblast cells with diSPIM. (a) XY maximum intensity projection from 351 time-point volumetric series. (b) XZ maximum intensity projection to accompany a. (c) Single XZ slice, highlighting membrane ruffling and infolding. Data are shown at 15-s intervals, but note that acquisition occurred every 5 s, demonstrating the capability of diSPIM to capture fast cellular events at high spatial resolution. (d) Long-term reorganization of the plasma membrane, indicating growth and remodeling of filopodial protrusions. These images were rendered using the ‘Hot Red’ lookup table in ImageJ. Scale bars, 5 μm. See also Supplementary Video 2 and Supplementary Data 8.