Rapid and gentle hydrogel encapsulation of living organisms enables long-term microscopy over multiple hours

Abstract

Imaging living organisms at high spatial resolution requires effective and innocuous immobilization. Long-term imaging places further demands on sample mounting with minimal perturbation of the organism. Here we present a simple, inexpensive method for rapid encapsulation of small animals of any developmental stage within a photo-crosslinked polyethylene glycol (PEG) hydrogel, gently restricting movement within their confined spaces. Immobilized animals maintain their original morphology in a hydrated environment compatible with chemical treatment, optical stimulation, and light-sheet microscopy. We demonstrate prolonged three-dimensional imaging of neural responses in the nematode Caenorhabditis elegans, recovery of viable organisms after 24 h, and imaging of larger squid hatchlings. We characterize a range of hydrogel and illumination conditions for immobilization quality, and identify paralytic-free conditions suitable for high-resolution single-cell imaging. Overall, PEG hydrogel encapsulation provides fast, versatile, and gentle mounting of small living organisms, from yeast to zebrafish, for continuous observation over hours.

Fig. 1

Mounting live C. elegans in hydrogels and on agarose pads for microscopy. a Schematic of worm mounting procedures by PEG hydrogel encapsulation or on agar pads. b–d Images taken over 9 h in a 10% PEG hydrogel (b) or on agarose pads with 25 mM sodium azide (c) or with 100 nm polystyrene beads (d). Red vertical line indicates a pharyngeal landmark position at each time point, blue lines indicate the initial landmark position, and white arrow indicates displacement relative to time t = 0 h. Arrowheads indicate morphological changes such as necrotic cell death after prolonged azide exposure. Scale bar, 10 µm. e Multiple larval and adult stages embedded in the same hydrogel. The hydrogel edge is indicated by a faint line (arrow), surrounded by about 100–200 µm of uncrosslinked polymer. Scale bar, 100 µm. f Worms embedded in a 20% PEG hydrogel were imaged immediately after crosslinking and after release 12 h later. Arrowheads indicate cavities in the hydrogel formerly occupied by worms. Scale bars, 200 and 100 µm (inset)

Fig. 2

Characterization of PEG hydrogel crosslinking rates. a Crosslinking time determined by immobilization of young adult animals in 10–20% PEG-DA exposed with 365, 312, and 308 nm ultraviolet sources. Each point represents the mean of n = 10 independent trials, with each trial averaging measurements from two to five worms. Error bars represent standard deviation. Statistics were performed using ordinary two-way ANOVA with Bonferroni’s post hoc tests for pairwise comparisons: *P < 0.001 for each concentration compared to 10% PEG-DA, and ⁺P < 0.001 or ⁽⁺⁾P < 0.05 for each source compared to 308 nm UV. b Absorbance spectrum of 0.001% I2959 (gray, left axis), and emission spectra for each ultraviolet exposure source (right axis). Emission spectra were normalized such that area under each curve matched total output power

Fig. 3

Buffer conditions before, during, and after hydrogel crosslinking influence immobilization for microscopy. Pre-exposure to hypo- or hyper-osmotic solutions for 10 min, or cooling pretreatment (snowflake symbol represents on ice or in a –20 °C freezer for 2 min), occurred in a droplet or microtube. Hydrogel solutions (20%) were prepared in water (H₂O), S-Basal buffer (SB), 25 mM sodium azide (azide) or 1 mM tetramisole (tet) in water, 500 mM glycerol in water, or 1.5× S-Basal buffer. a Each dot represents the mean movement index over 3 min (Supplementary Figs. 4, 5), n = 7–10 worms per condition. Vertical and error bars represent mean and standard deviation. b Images represent typical movement under the conditions indicated by black arrows. Arrowheads indicate the edge of the hydrogel; animal movement can occur within this confined space (white arrows). Scale bar, 30 μm. Statistics were performed using ordinary two-way ANOVA with Bonferroni’s post hoc tests for pairwise comparisons: *P < 0.0001 compared with the hydrogel control with SB, and ⁺P < 0.0001 compared with the hydrogel cooling control with SB

Fig. 4

Long-term volumetric imaging of optogenetically stimulated neurons in living C. elegans. Animals expressing Chrimson and GCaMP2.2b in AWA sensory neurons were stimulated by red light and imaged using a diSPIM light-sheet microscope. a Schematic of diSPIM objectives, hydrogel, and red light exposure. b Three-dimensional volumetric view of AWA neurons. c–e Maximum intensity projections highlighting ROIs of AWAL and AWR cell bodies and AWAR dendrite, at t = 0 h (c), 6 h (d), and 13.5 h (e) time points. Scale bars, 20 μm. f–h Time-lapse recordings of GCaMP signal in each ROI beginning at each time point. The stimulation LED was pulsed for 10 s, once per minute. Continuous recordings lasted for 1 h per time point; here, 30 min are shown for clarity. i–k Mean fluorescence (ΔF/F₀) is calculated for the 30 stimulation pulses indicated in f–h, with shading indicating s.e.m., and red bars above indicating 10-s red light exposure

Fig. 5

Encapsulation of a living pygmy squid hatchling in PEG hydrogel for light-sheet imaging. a Three-day-old squid hatchling was stained with 1 µM BODIPY C3 succinimidyl ester and embedded in a 1.2 mm thick, 4 mm diameter hydrogel disk and viewed in brightfield (a, b) and on a fluorescent dissecting microscope (c). d Raw light-sheet image slices of one squid arm indicated in panel c (box) are shown at 5 µm increments. Scale bars: 1 mm (a), 250 µm (b, c), 50 µm (d)