The overall goal of this procedure is to induce DNA damage in a sub-nuclear region of a cell using a confocal fluorescent microscopy laser. This method can help answer key questions of DNA repair such as how proteins are recruited and retained at sites of DNA damage. The main advantage of this technique is that it allows for real-time visualization of the protein of interest so the profile of recruitment and retention can be built.

Begin this procedure by placing a chambered slide containing the cells of interest in a stage top incubator maintained at 37 degrees Celsius and 5%carbon dioxide. Register image fields using an encoded automated microscope stage. Select a recognizable feature of the culture vessel such as the barrier between wells.

Collect an image and record the X-Y location. This will allow for alignment and registration of the X-Y locations of selected fields following sample preparation. Once image registration is complete, select a field for micro-irradiation and focus the sample.

For cells expressing fluorescently-labeled proteins, select the focal plane with the maximum nuclear cross-section in the fluorescent channel of interest. Focusing on the maximal cross-section of the nucleus is crucial because it ensures that the focal point is centered on the nucleus for monitoring recruitment of the protein of interest to the site of induced damage. Select a clear feature within the nucleus such as a nucleolus and move the focal plane up and down while observing this change in appearance.

The true focal plane will lie within the transition from light to dark. To focus the sample, select the focal plane in which the selected feature has the sharpest contrast. The next step is to register the position of the field of interest.

Create a three-by-three pixel square region of interest, or ROI, within the microscope software. Place this ROI over the nucleus of a cell to be damaged and set this ROI to be the damaged ROI. Collect a pre-damage image including the position of the damage ROI.

Acquire an image containing bright field in the florescence channel for the protein of interest. The cells are now ready for laser micro-irradiation. For this demonstration, the 405-nanometer laser will be used at 100%power.

The 405-nanometer laser dose is controlled by modulating the scan rate and performing one scan of the selected damage ROI. In this experiment, use eight and 0.5 frames per second for micro-irradiation at 405 nanometers. Selecting the appropriate laser power for damaging cells is critical to separating different DNA repair pathways.

Perform time-lapse image acquisition of bright field and florescence channels after damage. Adjust the duration and frequency of time-lapse to optimize data collection ideally capturing the accumulation of the fluorescent protein at the damage ROI and its dissociation over the time course of the experiment. After the time course is completed, select a new field of cells for damage and continue micro-irradiation and time-lapse imaging until the desired number of damaged cells is reached.

10 to 25 cells per selected condition is recommended. To increase the overall cell number for immunofluorescence analysis, damage additional fields within the culture vessel to generate a multi-field time course of the post-damage response. Record the X-Y location of each field and the time that the damage occurred.

Cells can be fixed immediately after damage or allowed to repair for selected increments of time before fixation. To begin this analysis, open the acquired images in an image analysis application such as NIS-Elements. For each cell to be measured, first generate a reference ROI that represents the nucleus.

Use a thresholding algorithm on the florescence signal that contains the pixels making up the nucleus, and then convert this area into a ROI. Next, create a six-by-six pixel ROI and place it over the damage ROI. This larger ROI is now the damage ROI for analysis.

For each cell to be measured, record the mean florescence intensity for the reference and damage ROIs. For each frame of the time course, adjust the reference ROI to ensure the nuclear area is accurately covered by the ROI and adjust the damage ROI to ensure that the damaged spot is covered. Record the mean fluorescence intensity of the fluorescent signal within each ROI for each frame of the time course.

For each cell, normalize the mean damage ROI fluorescence intensity to that of its corresponding reference ROI in each frame of the time-lapse. Here, the mean nuclear fluorescence intensity is used as the reference ROI and normalization is performed by subtracting the mean reference ROI fluorescence intensity from the mean damage ROI fluorescence intensity. Repeat the normalization for all damaged cells as well as for at least two undamaged control cells.

Finally, graph normalized intensity values over time to show changes in recruitment dynamics as a function of experimental treatment. Cells stably expressing XRCC1-GFP were irradiated and imaged before and after damage induction. Recruitment of XRCC1-GFP to the damage ROI was observed and the dynamics of recruitment measured.

The formation of double-strand breaks was examined using two markers. For both markers, the two second low-dose stimulation at 355 nanometers elicits no response at five minutes and 20 minutes and a weak and variable response at 10 minutes post-irradiation. The 10-second high-dose micro-irradiation at 355 nanometers induces an increased fluorescent signal within the damage ROI at five, 10 and 20 minutes post-irradiation that is reduced at 40 minutes.

These results indicate that the DNA double-strand break markers respond at 355-nanometer micro-irradiation in a dose-dependent manner. In contrast, 405-nanometer laser stimulation resulted in significant accumulation of both markers within the damage ROI regardless of the applied dose. Once mastered, this technique can be performed in about four hours for 10 cells if it is performed properly.

While performing this technique, it's important to tailor the laser wavelength and applied power to the repair process being monitored. Following this procedure, immunofluorescence can be performed to answer questions about recruitment of additional proteins or changes in phosphorylation status or other post-translational modifications. This technique allows researchers to explore dynamic recruitment of DNA repair proteins and better determine how protein domains and mutations affect the recognition of and response to DNA damage.

After watching this video, you should have a good understanding of how to use a laser-scanning confocal microscope to induce DNA damage and monitor recruitment of DNA repair proteins. Don't forget that working with lasers and chemicals such as formaldehyde can be extremely hazardous and precautions such as personal protective equipment should always be taken while performing this procedure.