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. 2018 Mar 6;13(3):e0193989.
doi: 10.1371/journal.pone.0193989. eCollection 2018.

Reversible and Long-Term Immobilization in a Hydrogel-Microbead Matrix for High-Resolution Imaging of Caenorhabditis Elegans and Other Small Organisms

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Free PMC article

Reversible and Long-Term Immobilization in a Hydrogel-Microbead Matrix for High-Resolution Imaging of Caenorhabditis Elegans and Other Small Organisms

Li Dong et al. PLoS One. .
Free PMC article

Abstract

The nematode Caenorhabditis elegans is an important model organism for biomedical research and genetic studies relevant to human biology and disease. Such studies are often based on high-resolution imaging of dynamic biological processes in the worm body tissues, requiring well-immobilized and physiologically active animals in order to avoid movement-related artifacts and to obtain meaningful biological information. However, existing immobilization methods employ the application of either anesthetics or servere physical constraints, by using glue or specific microfluidic on-chip mechanical structures, which in some cases may strongly affect physiological processes of the animals. Here, we immobilize C. elegans nematodes by taking advantage of a biocompatible and temperature-responsive hydrogel-microbead matrix. Our gel-based immobilization technique does not require a specific chip design and enables fast and reversible immobilization, thereby allowing successive imaging of the same single worm or of small worm populations at all development stages for several days. We successfully demonstrated the applicability of this method in challenging worm imaging contexts, in particular by applying it for high-resolution confocal imaging of the mitochondrial morphology in worm body wall muscle cells and for the long-term quantification of number and size of specific protein aggregates in different C. elegans neurodegenerative disease models. Our approach was also suitable for immobilizing other small organisms, such as the larvae of the fruit fly Drosophila melanogaster and the unicellular parasite Trypanosoma brucei. We anticipate that this versatile technique will significantly simplify biological assay-based longitudinal studies and long-term observation of small model organisms.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. C. elegans immobilization protocol using a hydrogel-microbead matrix.
(A) Worms are transferred from an agar plate into a droplet of liquid S Medium on a glass slide. (B) Precooled (~4°C) liquid Pluronic-microbead suspension is applied on the glass slide around the S Medium (red trace) and on a coverslip (not shown here). (C) The coverslip is positioned upside down and worms are immobilized in the gel-microbead matrix (red) in between the two glass substrates after thermalization (T ≈ 25°C). (D) Schematics of the worm immobilization technique with microbead spacers.
Fig 2
Fig 2. Characterization of different Pluronic (PF127) solutions.
(A) PF127 viscosity as a function of temperature for a range of concentrations, measured with a cone-plate viscometer. The viscosity curves show a sharp rise at a specific temperature, corresponding to the sol-gel transition. (B) The viscosity at 25°C follows a quadratic progression as a function of PF127 concentration. The sol-gel transition temperature decreases linearly with increasing Pluronic concentration.
Fig 3
Fig 3. The mechanism of C. elegans immobilization in the hydrogel-microbead matrix.
(A) Application of compression in the normal direction increases friction between the worm body and the glass surfaces. (1-ε) and (1+δ) denote the vertical and lateral deformation of a worm with a body diameter dw. (B) Bright field images of an adult hermaphrodite worm in a hydrogel-microbead matrix on a glass slide: freely moving (without coverslip), and in a compressed state with a coverslip. The vertical confinement is defined by the bead diameter. Arrows indicate the location where the lateral dorsoventral deformation of the worm body was measured. (C) Worm body extension (1+δ as a function of the degree of compression/confinement (1-ε for two different microbead spacers (40 μm and 30 μm). The range of values is determined by body diameter variations of the unsynchronized worm population used here. (**** p≤0.001, n = 15 for each group).
Fig 4
Fig 4. Evaluation of C. elegans immobilization and recovery for different conditions.
(A-F) Pairs of successive bright field images (10× objective) give an indication of adult worm motility under different conditions: (A-C) in buffer solution (one stroke intervals) and (D-F) in a PF127 (30% w/v) hydrogel matrix (using false colors to visualize two successive snapshots (5 s intervals). 3 different degrees of worm compression have been applied in either case: (A, D) no compression (no microbeads), and vertical confinement defined by 40 μm (B, E) or 30 μm (C, F) spacer microbeads, respectively. Rectangles indicate regions of interest for high-resolution imaging. Displacement amplitudes of 3 distinct worm body regions (sbc head close to the buccal cavity, sm mid-region and st tail), as indicated in (A) or (D), were measured. (G) Mean amplitude of displacement over 5 s intervals of the 3 worm body regions, in buffer solution or in PF127 under different conditions: no beads/no compression (nb), and with 40 μm or 30 μm spacer beads. Data is presented as mean±SD, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001 vs. nb in each condition, n = 15 for each group. (H) Fluorescent image of mitochondria aggregates in a Pmyo-3::mito::GFP worm immobilized in a PF127-microbead (30 μm) matrix. A selected mitochondrial structure in mid-body region close to vulva is indicated by crosshairs (63× objective). (I) Mean absolute displacement over 5 s intervals of different selected mitochondrial structures near terminal bulb (stb), vulva in the mid-body region (sm) and anus (st). Animals were immobilized in a PF127-microbead (30 μm) matrix. Data is presented as mean±SD, n = 10 for each group. (J) Frequency of repeating motion patterns for freely moving adult worms (thrashes in buffer) and during immobilization under different conditions in PF127 (mainly forward-backward motion). Data is presented as mean±SD, **** p≤ 0.0001 vs buffer group, n = 15 for each group. (K) Evaluation of worm recovery from short-term (10 min, orange bars) and long-term (1 hour, blue bars) immobilization in PF127 (30% w/v) and tetramisole (1mM). The thrashing frequency was measured 10 min, 3 h and 1 day after release, respectively. The control population was maintained in buffer solution (no immobilization) all the time (ctl, grey bar). Data is presented as mean±SD; for short-term immobilization, ***p≤ 0.001 vs ctl group; for long-term immobilization, *p≤ 0.05 and ****p≤ 0.0001 vs ctl group. N = 15 for each group. (L) Evaluation of worm fertility (N2 wild type) after long-term immobilization (1 hour and 2 hours) in PF127 (30% w/v) and using tetramisole (1mM, 1 hour) at 20°C. Measurements were made at adult stage at day 1, 2 or 3 after release. Data is presented as mean±SD, *p≤ 0.05 and **p≤ 0.01 vs ctl group of each day, n = 10 for each group.
Fig 5
Fig 5. High-resolution confocal imaging of C. elegans.
(A) Schematics of an immobilized adult worm (side view) using two different techniques, i.e. tetramisole-induced worm paralysis (current gold standard) and our gel-based method (dashed rectangles indicate the location of the imaging areas). (B) Representative confocal fluorescent images of dynamic alterations of mitochondrial networks in C. elegans body wall muscle cells during continuous immobilization up to 3 hours, visualized using the Pmyo-3::mito::GFP reporter. Images are taken through a 63× NA 1.4 oil immersion objective using the standard immobilization method (left panels) and our technique (right panels). Scale bars = 5 μm.
Fig 6
Fig 6. Longitudinal analysis of mitochondrial morphology in the body wall muscle cells of C. elegans worms under different RNAi conditions.
In wild-type control worms (empty vector), the mitochondrial network is well interconnected on day 1, but begins to fragment with age on day 3 (inset A). fzo-1 knockdown inhibits mitochondrial fusion and produces many fragmented mitochondria patterns (fission, inset B), whereas drp-1 RNAi knockdown inhibits division and produces larger aggregated networks (fusion, inset C). Images are representative of n = 4–6 worms analyzed per condition, visualized via confocal microscopy (63× NA 1.4 oil immersion objective) using the Pmyo-3::mito::GFP reporter. Scale bars = 5 μm. Insets A, B and C are digital zooms (x3.6) of the indicated circular areas.
Fig 7
Fig 7. Protein aggregate morphology and progression in different C. elegans neurodegenerative disease models.
(A) Time-lapse bright field and fluorescent wide field images of an AM140 worm (Huntington’s disease model) immobilized in the gel-microbead matrix. PolyQ35 proteins exhibit a transition from a soluble state to an aggregated form as the worm ages from day 3 to day 7 of adulthood (10× objective, scale bar = 100 μm). (B) Time-lapse fluorescent pictures of an AM725 worm (Amyotrophic Lateral Sclerosis model) displaying aggregated SOD1 proteins throughout its adulthood (day 2 to day 7), with aggregate size growing over time (10× objective, scale bar = 100 μm). (C) Fluorescent images of α-syn aggregates in a NL5901 worm (Parkinson’s disease model), allowing accurate visualization of smaller aggregates (20× and 50× objectives, scale bars = 50 μm and 20 μm, respectively). (D-E) Temporal evolution (from day 2 or 3 to day 7) of the average aggregate size, number of aggregates and total aggregate area per worm, in (D) AM140 and (E) AM725 worms. Data is presented as mean+SEM, ***p≤0.001 and ****p≤0.0001, n = 15–20 for each group).
Fig 8
Fig 8. Gel-based immobilization of other small organisms.
(A) Image of an immobilized D. melanogaster 1st instar larva (length ~2 mm). (B) Image of an immobilized T. brucei unicellular parasite (length ~20 μm). (C) Pluronic hydrogel concentration-related reduction of head thrashes for D. melanogaster 1st instar larvae, D. melanogaster larvae in the prepupal stage (n = 15 for each group), and reduction of body thrashes (swimming frequency) for T. brucei (n = 20). Data is present as mean±SD, * p≤0.05, ** p≤0.01, *** p≤0.001 and **** p≤0.0001 vs each 0% w/v Pluronic group, n = 15–20 for each group.

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This work is supported by ERC-2012-AdG-320404 (http://cordis.europa.eu/project/rcn/106969_en.html). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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