Inverted selective plane illumination microscopy (iSPIM) enables coupled cell identity lineaging and neurodevelopmental imaging in Caenorhabditis elegans

Abstract

The Caenorhabditis elegans embryo is a powerful model for studying neural development, but conventional imaging methods are either too slow or phototoxic to take full advantage of this system. To solve these problems, we developed an inverted selective plane illumination microscopy (iSPIM) module for noninvasive high-speed volumetric imaging of living samples. iSPIM is designed as a straightforward add-on to an inverted microscope, permitting conventional mounting of specimens and facilitating SPIM use by development and neurobiology laboratories. iSPIM offers a volumetric imaging rate 30× faster than currently used technologies, such as spinning-disk confocal microscopy, at comparable signal-to-noise ratio. This increased imaging speed allows us to continuously monitor the development of C, elegans embryos, scanning volumes every 2 s for the 14-h period of embryogenesis with no detectable phototoxicity. Collecting ∼25,000 volumes over the entirety of embryogenesis enabled in toto visualization of positions and identities of cell nuclei. By merging two-color iSPIM with automated lineaging techniques we realized two goals: (i) identification of neurons expressing the transcription factor CEH-10/Chx10 and (ii) visualization of their neurodevelopmental dynamics. We found that canal-associated neurons use somal translocation and amoeboid movement as they migrate to their final position in the embryo. We also visualized axon guidance and growth cone dynamics as neurons circumnavigate the nerve ring and reach their targets in the embryo. The high-speed volumetric imaging rate of iSPIM effectively eliminates motion blur from embryo movement inside the egg case, allowing characterization of dynamic neurodevelopmental events that were previously inaccessible.

Fig. 1.

iSPIM, plane illumination on an inverted microscope base. Two water-immersion objectives are mounted onto a Z translation stage that is bolted directly onto the illumination pillar of an inverted microscope. The demagnified image of a rectangular slit (MASK) is reimaged with lenses (L) and excitation iSPIM objective (EXC OBJ), thus producing a light sheet at the sample (S). For clarity, relay lens pairs between L and EXC OBJ are omitted in this schematic, but are included in Fig. S1. Sample fluorescence is detected (DET OBJ) using appropriate mirrors (M), emission filters (F), lenses (L), and camera. EXC OBJ is fixed in place and the light sheet is scanned through the sample using a galvanometric mirror (not shown). A piezoelectric objective stage (PS) moves DET OBJ in sync with the light sheet, ensuring that detection and excitation planes are coincident. The sample S is mounted onto a coverslip (C) that is placed onto a 3D translation stage, thus ensuring correct placement of S relative to iSPIM objectives. S may also be viewed through objectives (CONV OBJ), dichroic mirrors (DM), and optics in the conventional light path of the inverted microscope.

Fig. 2.

High-speed nuclear imaging in the nematode embryo. Embryos with GFP-histones were volumetrically imaged at 30 vol/min from the two-cell stage until hatching. (A) Selected maximum-intensity projections from a representative embryo, highlighting different developmental stages (Movie S2). (Scale bar: 5 μm.) (B) Highlighted nuclei (circled in red, A Top Right) comparing maximum-intensity projections between iSPIM (Left, images sampled every 2 s but shown every 6 s due to space constraints) and spinning-disk confocal microscopy (Right, images sampled every minute, taken on a different embryo).

Fig. 3.

Dual-color iSPIM and lineaging identifies neuronal cells expressing CEH-10/Chx10. (A) DIC and iSPIM maximum-intensity projections at indicated time points, highlighting ubiquitously expressed histone:mcherry (red) and ceh-10p:GFP (green) at selected times points. iSPIM images are from a representative embryo; DIC pictures were taken on a different animal and are provided only as a reference (Movie S3). (B) Cartoon model to accompany A, also emphasizing location of neurons with respect to the anatomical body plan of the embryo. (C) Computer-derived lineage tree for the identified neurons in A.

Fig. 4.

Neuronal morphology and migration through early twitching. (A) Selected maximum-intensity projections of ceh-10p:GFP, highlighting migration of neurons after expression of GFP, through onset of early twitching. The outline of the embryo is indicated by the dotted line. (B) Outlines of the migratory path of individual neurons before embryonic twitching. (C) Migration kinetics of CANs from the dataset shown in A, through early twitching. 3D displacements are measured from the anterior tip of the embryo. (D) An example of somal translocation before twitching from a dual-color iSPIM dataset. (E) Higher-magnification view of the blue boxed region in A, showing amoeboid movement of CANs posttwitching as neurons migrate posteriorly. Dual-color images in D were acquired from a representative embryo at 12 vol/min; single-color images for other panels were acquired from another representative embryo at 10 vol/min (Movie S4). In all images, anterior is Left and posterior Right.

Fig. 5.

Neuronal outgrowth through twitching. (A) Selected maximum-intensity projections emphasizing ALA neurite outgrowth from a representative embryo. The outline of the embryo is indicated by the dotted line. The red boxed region highlights ALA (Movie S5). (B) Higher-magnification view. The red dot marks ALA soma; the green star marks the Left neurite outgrowth. (C) Selected projections emphasizing CAN neurite outgrowth, from the same embryo as in A and B (Movie S6). Purple arrows highlight direction of outgrowth. Note that as the embryo is twitching inside the egg case, the relative position of the CANs inside the embryos changes between volumes. High-speed volumetric imaging through iSPIM enabled visualization of outgrowth through twitching with no motion blur. (D) Higher-magnification view, emphasizing growth cones (orange stars). (E) Quantification of anterior extensions (distance from center of cell body to tip of axon) over time, measured from four animals. Data were acquired at 30 vol/min. (F) Higher-magnification view of outgrowth of both anterior (pink arrows) and posterior (red arrows) CAN neurites, from the same embryo as in A–D. The posterior outgrowth became visible ∼12–13 hpf.