zWEDGI: Wounding and Entrapment Device for Imaging Live Zebrafish Larvae

Abstract

Zebrafish, an established model organism in developmental biology, is also a valuable tool for imaging wound healing in space and time with cellular resolution. However, long-term imaging of wound healing poses technical challenges as wound healing occurs over multiple temporal scales. The traditional strategy of larval encapsulation in agarose successfully limits sample movement but impedes larval development and tissue regrowth and is therefore not amenable to long-term imaging of wound healing. To overcome this challenge, we engineered a functionally compartmentalized device, the zebrafish Wounding and Entrapment Device for Growth and Imaging (zWEDGI), to orient larvae for high-resolution microscopy, including confocal and second harmonic generation (SHG), while allowing unrestrained tail development and regrowth. In this device, larval viability was maintained and tail regrowth was improved over embedding in agarose. The quality of tail fiber SHG images collected from larvae in the device was similar to fixed samples but provided the benefit of time lapse data collection. Furthermore, we show that this device was amenable to long-term (>24 h) confocal microscopy of the caudal fin. Finally, the zWEDGI was designed and fabricated using readily available techniques so that it can be easily modified for diverse experimental imaging protocols.

Keywords: device; live imaging; microscopy; wounding.

FIG. 1.

Design and implementation of zWEDGI. (A) Labeled and dimensioned enface SolidWorks drawing of the zWEDGI mold. (B) SolidWorks isometric of mold and PDMS device. Upper expanded view details the structure of the restraining tunnel and lower view shows positioning of larva. All dimensions are in mm unless specifically noted. (C) Flow chart illustrating procedure for making zWEDGI. Details of the procedure are in the Materials and Methods section. (D) Finished PDMS device plasma bonded to glass bottom dish and a detailed view of a single channel with compartments labeled. Scale bar = 1 mm. PDMS, polydimethylsiloxane; zWEDGI, zebrafish Wounding and Entrapment Device for Growth and Imaging.

FIG. 2.

Positioning of larva in channel. (A) Dissecting microscope images showing 2, 3, and 4 dpf fixed larvae loaded into zWEDGI channel, with enlargement to show detail. (B) A minimal amount of agarose, labeled with rhodamine 6G and filling the loading chamber, exits from restraining tunnel into the wounding chamber, as detected by the pink halo (arrow). Residual agarose can be removed using a syringe needle with minimal impact on the tail, leaving the tail unrestrained in buffer in the wounding chamber. (C) Semicircular wounding chamber is designed to accommodate scalpel blade (arrow) for caudal fin transection. Scale bar = 1 mm. dpf, days postfertilization.

FIG. 3.

Growth and caudal fin regrowth are maintained in the zWEDGI. (A) Images from a dissecting microscope showing larvae collected within 30 min of wounding (0 dpw), 1 and 2 dpw that were anesthetized and maintained in E3 buffer (unrestrained), 1% agarose or zWEDGI with 1% agarose over head region and tail unrestrained in buffer. Scale bar = 1 mm. (B) Graph showing body length (eye to edge of tail fin) in the three mounting conditions. (C) Graph comparing regrowth (regenerate length) of transected tail fin with the three mounting conditions. Data normalized to mean of 0 dpw for a given treatment, n ≥ 28 larvae per treatment over a total of three replicates. **p < 0.01, ***p < 0.0001. dpw, days post-wound.

FIG. 4.

High-resolution light microscopy of larval tails in zWEDGI. (A) Z-projections of SHG images collected with similar imaging parameters of a fixed sample (single time point) and a live sample mounted in a zWEDGI (multiple time points), demonstrating short time frame imaging immediately after wounding. (B) Confocal long-term time lapse of tail development showing gfp-tagged neuron growth. (C) Dissecting microscope image with accompanying multiphoton image showing isolation of rhodamine 6G (red) to the wounding chamber, 2 h after initial application of the dye to the wounding chamber, illustrating only minor infiltration of dye into the restraining tunnel and levels below detection in the head region. (A, B) Scale bar = 100 μm. (C) Scale bar = 1 mm. SHG, second harmonic generation.