Bx51 widefield 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.