Sp5 inverted confocal fluorescence microscope

To provide a nonadherent coating for cell patterning, 200 μL of 3% (w/v) molten UltraPure Agarose 1000 (Thermo Fisher Scientific) was added to the central cavity of an acoustic patterning device and allowed to gel by cooling to room temperature. A suspension of fixed, stained chondrocytes was added to an equivalent volume of molten type VII-A agarose, with both solutions mixed at 37 °C. The final cell density was 3 × 10⁷ cells mL^–1 and the final agarose concentration was 1.5% (w/v). 200 μL of this solution was added to the central cavity of the acoustic patterning device and a single piezo-transducer pair was driven at a frequency of 6.7 MHz and an amplitude of 20 V_pp. The acoustic field was maintained for 3 min, during which time the solution was cooling to room temperature. After 1 h, the patterned agarose hydrogel was removed from the device for imaging. Low magnification brightfield images were captured with an Olympus BX51 widefield microscope, while high magnification images were obtained using a Leica SP5 inverted confocal fluorescence microscope.

The stab wound model was verified by F-actin staining, using a protocol adapted from Goody et al.⁵⁸ , and performed on a separate set of embryos to those used in the Raman scans with different embryos used for each timepoint. The wounded embryos were fixed overnight in 4% (w/v) PFA at 4 °C. The next day, the embryos were washed 3 × 5 min in 0.5 mL 0.1% (v/v) Tween 20 (Sigma) in PBS (Gibco) and then washed for 90 min at room temperature in 0.5 mL 2% (v/v) Triton X-100 (Sigma) in PBS (Gibco). The Triton X-100 solution was then replaced with 19 µL of fresh 2% (v/v) Triton X-100 with 1 µL of rhodamine phalloidin (Thermo Fisher). Staining was performed overnight at 4 °C with gentle shaking. Prior to imaging, the embryos were gradually transferred to 80% (v/v) glycerol through subsequent washing steps of increasing glycerol concentration (20, 40, 60, 80%). The embryos were then mounted on a glass bottomed petri dish (Cellview Cell Culture Dish, PS 35/10 MM, glass Bottom, Greiner Bio-One) and then imaged using an SP5 inverted confocal fluorescence microscope (Leica) with a 20x/0.5 PL FLUOTAR objective lens (Leica).

A suspension of fixed and stained chondrocytes in PBS was added to the central cavity of the acoustic patterning device and imaged using a Leica SP5 inverted confocal fluorescence microscope, with images captured every 1 s. After 5 s, the cells were subjected to an ultrasound standing wave (6.7 MHz, 9 V_pp) with images continuing to be captured every 1 s. ImageJ software (National Institutes of Health, USA) was used to export images from time-lapse stacks and perform Fast Fourier Transform and profile plot analyses for the 20 s following ultrasound field exposure.