This article describes a method for extracting bacterial colonies from a photodegradable hydrogel using UV light exposure. The process involves careful monitoring and adjustment of light patterns to minimize damage to the cells while facilitating their release for further analysis.
Take a coverslip with a photodegradable hydrogel layer that contains entrapped bacterial colonies, genetically modified to emit fluorescence.
Place the coverslip into a holder.
Add media to prevent sample dehydration.
Under a brightfield microscope, focus on a single colony of interest.
Using appropriate software, design a light pattern for cell extraction at the colony periphery to break down the structural bonds of the hydrogel and minimize UV exposure to the cells.
Adjust the UV light intensity and exposure duration.
Initiate UV light exposure to break the structural bonds in the hydrogel at the exposed areas that facilitate the release of the target colony.
Using a fluorescence filter, identify the colony.
Place a tubing tip at the spot, with the other end connected to a syringe.
Switch to brightfield mode and, using the syringe, aspirate the released colony.
Transfer the colony into a tube for further analysis.
Place the sample in a PDMS holder and add the defined media on top of the sample to prevent sample dehydration and provide a carrier solution for released cells. After bringing the calibration mirror in position, adjust the microscope focus to get a sharp image of the colonies within the hydrogel or microwell array and inspect to identify colonies or wells of interest.
Here, design the light patterns while the camera view shows the colonies inside the sample to test different patterns for cell extraction and save the defined pattern. Then select the session control section, add the saved sequence under the tab titled with the patterned illumination tool product name. Choose the option for stimulating the pattern to view and adjust the desired exposure location.
Next, adjust the light intensity to 60% and the exposure time to 40 seconds under the LED control tab and start the exposure process. Monitor the hydrogel degradation in real time and Brightfield mode to ensure the cell release. To collect the released cell, change the microscope filter from Brightfield to TRITC to visualize the exposed area of the sample by the naked eye.
Once the exposed area is located, place the end of the tubing upon the irradiated spot, then change the microscope filter back to Brightfield to monitor cell retrieval in real time. Use the syringe attached to the other end of the tubing to carefully withdraw 200 microliters of solution containing the released cells and transfer the solution into a 1.5 millimeter centrifuge tube for DNA analysis or plating
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