Spatially isotropic four-dimensional imaging with dual-view plane illumination microscopy

Abstract

Optimal four-dimensional imaging requires high spatial resolution in all dimensions, high speed and minimal photobleaching and damage. We developed a dual-view, plane illumination microscope with improved spatiotemporal resolution by switching illumination and detection between two perpendicular objectives in an alternating duty cycle. Computationally fusing the resulting volumetric views provides an isotropic resolution of 330 nm. As the sample is stationary and only two views are required, we achieve an imaging speed of 200 images/s (i.e., 0.5 s for a 50-plane volume). Unlike spinning-disk confocal or Bessel beam methods, which illuminate the sample outside the focal plane, we maintain high spatiotemporal resolution over hundreds of volumes with negligible photobleaching. To illustrate the ability of our method to study biological systems that require high-speed volumetric visualization and/or low photobleaching, we describe microtubule tracking in live cells, nuclear imaging over 14 h during nematode embryogenesis and imaging of neural wiring during Caenorhabditis elegans brain development over 5 h.

Figure 1

Dual-view iSPIM setup. 0.8 NA water-immersion objectives (A/B) are mounted orthogonally onto a z translation stage that is bolted directly onto the illumination pillar of an inverted microscope. In conjunction with other optics (Supplementary Fig. 1), both objectives produce a light sheet at the sample. Excitation A(B) occurs via objective A(B), and the resulting fluorescence is collected through perpendicular objective B(A), and imaged onto camera B(A) by means of dichroic mirrors, emission filters and lenses. Excitation (blue) and detection (red) are shown occurring simultaneously along both light paths in the lower schematic, but in reality volumetric imaging occurs sequentially as shown in the upper right inset. During acquisition, sample and objective A(B) are held stationary, the light sheet is scanned through the sample using galvanometric mirrors (not shown), and a piezoelectric objective stage moves objective B(A) in sync with the light sheet, ensuring that excitation and/or detection planes are coincident. The sample is mounted onto a rectangular coverslip that is placed onto a 3D translation stage, ensuring correct placement relative to objectives. The sample may also be viewed through objective C (see upper left inset), dichroic mirror, emission filter, lens and camera C placed in the conventional light path of the inverted microscope. This objective is particularly useful in finding or screening samples.

Figure 2

Improving resolution isotropy with different fusion schemes. (a–e) Image planes from the center of volumetric data sets of 100-nm fluorescent beads, for single views (a), deconvolved single views (b), arithmetic fusion (c), deconvolved arithmetic fusion (d) and joint deconvolution (e). (f) Comparison of axial and lateral line profiles from (a,b,e). See also Supplementary Table 1 and Supplementary Video 1. xy: lateral view; zy; axial view.

Figure 3

Bleaching comparison between SDCM and diSPIM. (a) GFP-EB3 cells were imaged with SDCM (top row) and diSPIM (bottom row), at equivalent illumination dose. Cells imaged with SDCM were temporally sampled 3× less frequently than diSPIM (3× fewer total volumes, also 3.2× fewer planes per volume), but exhibited significantly more bleaching. (b) Higher magnification views of the green rectangular subregions in a. (c) The bleaching rate was quantified from a 20 × 20 μm² area inside each cell; signal-to-noise ratio (SNR) was calculated as the ratio of the averaged intensity of this area over the s.d. of the background in an identical area outside the cell. Data were fitted to single exponentials; half-time to complete bleaching is indicated on the graph. See also Supplementary Video 2.

Figure 4

Dual-view iSPIM enables microtubule tracking in 4D. (a) xy, zy and xz maximum-intensity projections from a single time point taken from a 5-min volumetric series, showing GFP-EB3–labeled microtubules in human umbilical vein cells, on coverslips (a thin cell, left column, and a thicker cell, middle) and inside a collagen gel (right). Inset indicates geometry of figure axes relative to the light sheet propagation axes. See also Supplementary Video 3. (b) Higher magnification views of the green rectangular subregions in a, projected over the indicated time interval and showing example tracks in red. (c) Quantification of average speed (left graph) and lifetime (right graph) as a function of angle relative to coverslip for the different samples. Mean and s.d. are shown for N = 75, 75 and 100 tracked tips (from three thin cells on coverslips, two thick cells on coverslips and three cells in gels) on the right-hand portion of each graph.

Figure 5

Dual-view iSPIM improves axial resolution in 4D embryonic imaging. (a) Selected diSPIM maximum-intensity projections of GFP-labeled histones in nematode embryo, from 786 time-point volumetric series. Projections were computed 60 degrees relative to y axis. (b) Comparison between diSPIM (left column), single view iSPIM (middle) and SDCM (right) at the same time point in embryogenesis. Lower two rows: higher magnification views of boxed nuclei in top two rows. All times are hours:minutes post fertilization. Projections are taken at 0 or 90 degrees relative to y axis. See also Supplementary Videos 4,5 and Supplementary Figure 11.

Figure 6

Spatiotemporal dissection of AIY neurite outgrowth. (a) AIY neurons in the context of the developing nematode embryo. Outline indicates eggshell. (b) Top: AIY morphology in the adult nematode embryo, with zone 1 and zone 3 regions marked. Bottom: length of zones 1 and 3 during AIY development. Time intervals A–D are introduced to clarify description in text. The blank region (dashed lines in interval D) arises due to lowered expression of GFP during this period, probably due to the characteristics of the promoter region used. Data: mean ± s.d., from four embryos. (c) Maximum intensity projections from iSPIM (left column) and diSPIM (middle) at indicated times (and rotation angle relative to y axis). Volumes are taken from a 910 time-point series obtained from a single animal. Note the increased resolution of diSPIM better resolves fine neurites and cell bodies as compared to iSPIM. Red stars and pink arrows indicate the position of the AIY cell bodies and neurites, respectively. Right column: accompanying cartoon. Scale bars, 5 μm. All times are hours: minutes:seconds post fertilization. See also Supplementary Videos 7 and 8.