Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

1. Cell Culture and generation of stable cells

1. Grow CHO-K1 cells in minimal essential medium supplemented with 10% fetal bovine serum. Maintain cells in a humidified incubator with 5% CO₂ at 37 °C.
2. Transfect CHO-K1 cells with 1 µg of plasmid DNA containing human XRCC1 with a C-terminal green fluorescent protein (GFP) tag using a commercial transfection reagent following the manufacturer's instructions.
3. Select CHO-K1 cells stably expressing the human XRCC1-GFP fusion using 800 µg/mL geneticin and enrich the XRCC1-GFP expressing population using fluorescence assisted cell sorting (FACS).
4. Plate cells to be micro-irradiated in culture vessels with coverglass bottoms, so they reach approximately 75% or greater confluency on the following day. Some spaces between cells are desirable, particularly if using registration to return to the same image field (described in section 3.2).

2. Microscope set-up

1. Select a laser scanning confocal microscope equipped with suitable lasers and optics, and control software for micro-irradiation/photostimulation. Presented experiments use a laser scanning confocal microscope that has been modified to include a 355 nm laser, fiber-coupled to a galvanometer photoactivation miniscanner and controlled using the manufacturer's control and analysis software. This system can perform micro-irradiation in a user-designated region of interest (ROI) using the photoactivation miniscanner (355 nm laser) or the standard confocal galvanometer (405 nm laser).
2. Select the wavelength to be used for micro-irradiation, ensuring that all optical components in the micro-irradiation lightpath are suitable for the chosen wavelength.
   NOTE: The presented system uses a 40× C-Apochromat (numerical aperture (NA) 1.2) oil immersion objective together with a UV filter cube for 355 nm micro-irradiation, and a 20× C-Apochromat (NA 0.75) dry objective using the standard confocal lightpath for 405 nm micro-irradiation. The 20× objective used in the setup is not compatible with the 355 nm wavelength; therefore we used the 40× for 355 nm only. Using the dry objective for damaging allows immunofluorescence protocols to be applied downstream without cleaning off immersion oil, which is why we utilize this objective when possible.
3. Configure the microscope for the micro-irradiation experiment. To maintain consistency between experiments, designate a standardized configuration of the microscope components to be used for each type of micro-irradiation. Save these settings as a preset within the microscope's operating software, if possible.
   NOTE: In the presented system, cells are micro-irradiated and imaged using unidirectional scanning, a scan resolution of 1024×1024 pixels with 1x scan zoom, at a frame rate of 8 frames per second (fps), and with the pinhole set to approximately 4 Airy Units (AU), as determined for the 488 nm laser (69 µm). This open pinhole setting was chosen to maximize the amount of light captured in each image, allowing the use of lower imaging laser powers and reducing photobleaching.

3. Laser micro-irradiation

1. Place the prepared culture dish, containing cells of interest, onto the microscope stage and lock securely into place.
   1. If available, place chambered slide in a stage-top incubator and maintain at 37 °C with 5% CO₂ during damage induction. This step is most important for live-cell timelapse imaging or long imaging sessions. Otherwise place cells on the microscope stage and perform irradiation at room temperature (~ 25 ˚C), and then quickly return cells to an incubator at 37 °C with 5% CO₂.
2. Register image fields to allow the researcher to return to the same location after performing fixation, staining, or other procedures. Perform registration of damaged fields using an encoded, automated microscope stage or cell culture coverglass vessels with an etched or labeled grid of XY locations.
   1. If using software image registration, select a recognizable feature of the culture vessel (i.e., the corner of the coverglass or barrier between wells), collect an image, and record the XY location. This will allow for alignment and registration of the XY locations of selected fields following sample preparation.
   2. If using manual registration, record grid or etched locations for each field manually, being careful to record the orientation and placement of the dish on the stage.
   3. If no identifying features for the cell culture vessels are available, identify damaged fields by visual inspection of cell morphology and density. Take care to select fields with sufficiently distinct features to be recognizable during later imaging.
      NOTE: This can be time consuming to perform unless the orientation and area of the culture vessel is tightly restricted.
3. Select a field for micro-irradiation and focus the sample. For cells expressing fluorescently labeled proteins, select the focal plane with the maximal nuclear cross-section in the fluorescent channel of interest. For cells not expressing labeled proteins, or for those without clear nuclear localization, phase contrast or differential interference contrast (DIC) imaging can be used to find the correct focal plane.
   ​NOTE: A useful feature of phase contrast and DIC imaging is the fact that as the focal plane moves through the sample, the same feature can appear light or dark depending on the relative focal plane.
   1. 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.
4. Register the position of the field of interest. Create a 3×3 pixel square ROI within the microscope software, place this ROI over the nucleus of a cell to be damaged, and set this ROI to be the damage ROI.
5. Collect a pre-damage image, including the position of the damage ROI.
   1. For experiments that do not use fluorescent proteins, acquire a phase-contrast or differential interference contrast (DIC) brightfield image to identify and record nuclei for damage.
   2. For live-cell experiments using fluorescent proteins, acquire an image containing brightfield and the fluorescence channel for the protein of interest (i.e., laser line 488 nm for excitation of GFP, laser line 561 nm for excitation of RFP). In experiments using the CHO-K1 XRCC1-GFP, fluorescence excited by the 488 nm laser line was simultaneously collected with the transmitted DIC brightfield channel.
6. Initiate laser damage.
   1. In the presented system, control the laser dose by the total time spent scanning the damage ROI. Because the galvanometer for the 355 nm laser operates at a fixed scan rate, laser dose is controlled by the time spent repeatedly scanning the selected damage ROI: in the data presented here, micro-irradiation at 355 nm is performed for 2 and 10 seconds.
   2. In contrast, control the 405 nm laser dose by modulating the scan rate and perform one scan of the selected damage ROI. In the data presented here, use 8 and 0.5 fps for micro-irradiation at 405 nm. A scan rate of 8 fps delivers a lower laser dose than 0.5 fps, because the laser spends less time on each pixel during the scan. Both lasers are operated at 100% power. See section 3.7 for instructions on measuring laser power directly.
      NOTE: Each individual microscope system may differ in how laser power is delivered to a designated ROI, similar to how the 355 nm and 405 nm differ in the presented system. Users will need to determine this procedure for their microscope system and report these parameters within their methods sections.
   3. For live cell imaging, perform timelapse image acquisition of brightfield and fluorescence channels. Adjust duration and frequency of timelapse 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. Experiments using XRCC1-GFP collected images every 30 seconds for 20 minutes.
      ​NOTE: The frequency of the timelapse imaging can be limited by photobleaching of the fluorophore, complicating analysis of accumulation and dissociation. Photobleaching can be evaluated by observing undamaged cells during the timelapse and adjusting acquisition conditions to minimize fluorescent signal loss in these undamaged cells. Some photobleaching may be unavoidable, so users may compensate for the signal loss by normalizing the fluorescent intensities of damaged cells to those of undamaged cells in each frame of the timelapse.
      1. After the time course is completed, select a new field of cells for damage or fix damaged cells for further analysis, as described in section 4.
      2. Continue micro-irradiation and timelapse imaging until the desired number of damaged cells is reached.
         ​NOTE: We recommend damaging a total of 10-25 cells per selected condition in order to assess the cell-to-cell heterogeneity in response to the induced damage. Although most confocal systems will allow users to damage more than one cell during each micro-irradiation, this introduces staggered damage initiation times, which can complicate analysis of peak recruitment time over a large number of cells or dissociation time for fast events. In some systems, simultaneous damage induction over several ROIs may reduce the laser dose received by each independent ROI. Therefore, unless these timing/power issues are directly addressed by the user, it is recommended that cells be damaged individually.
   4. For analysis by immunofluorescence (IF), perform damage and either immediately fix cells, as described in section 4, or allow cells to repair for selected increments of time (i.e., 1, 5, 10 or 20 min). After the desired time elapses, fix and stain the cells, as detailed in section 4.
      1. To increase overall cell number for IF analysis, damage additional fields within the culture vessel to generate a multi-field time course of the post-damage response. Record the XY location of each field and the time that the damage occurred. After the desired total repair time elapses, fix and stain the cells as detailed below.
7. After observing of protein recruitment at selected doses, measure and report laser power levels post-fiber and post-objective to accurately characterize and report laser doses. We use a compact digital power meter and two different photodiode sensor heads; one coupled directly to the laser fiber to measure post-fiber output, and the other placed on the microscope stage to measure post-objective output.
   1. To measure laser power, place the sensor in the desired configuration (post-fiber or post-objective) and perform micro-irradiation as described above, while recording the measurements made by the power meter. Be aware that the sampling rate of the power meter may not be fast enough to capture very rapid micro-irradiation events, so it may be necessary to run multiple iterations of the experiment to ensure that the power meter accurately detects the applied dose.
      NOTE: For the 355 nm laser, we measured an average peak power of approximately 5 mW post-fiber and approximately 19 µW post-objective. For the 405 nm laser, micro-irradiation was performed in loops of 30 iterations to overcome the 0.01 s maximum sampling rate of the power meter, and average peak powers of 1.5 mW and 2.4 mW were measured using scan speeds of 8 and 0.5 frames per second, respectively. Every power meter is different. Users will need to determine the operational wavelengths and sampling rates for their individual power meter.

4. Immunofluorescence staining procedures

1. Analyze strand break induction by IF staining.
   1. Fix cells with 3.7% formaldehyde (CAUTION) in phosphate buffered saline (PBS) for 10 min. Aspirate formaldehyde solution and wash 3 times with PBS. The protocol can be stopped here by placing PBS back on the cells, and the cells can be stored at 4 ˚C for up to 1 week.
      CAUTION: Formaldehyde is toxic and carcinogenic. Wear proper personal protective equipment and dispose of the toxic substance as instructed by institutional environmental health and safety procedures.
   2. Permeabilize cells using 0.25% Triton X-100 in PBS for 10 min at room temperature (RT) and then wash 3 times with PBS.
   3. Block non-specific antibody binding by incubating for 30 min in PBS containing 1% bovine serum albumin (BSA) at RT.
   4. Incubate with primary monoclonal antibody against γH2AX and primary polyclonal antibody against 53BP-1 both diluted 1:750 in PBS containing 1% BSA for 1 h at RT, and then wash 3 times with PBS.
   5. Incubate cells with Alexa 488 goat anti-mouse and Alexa 546 goat anti-rabbit both diluted 1:2000 in PBS containing 1% BSA for 1 h at RT, and then wash 3 times with PBS.
   6. Stain nuclear DNA with a 10 mg/mL solution of DAPI (4',6-Diamidino-2-Phenylindole, CAUTION) diluted to 1:5000 in PBS for 5 min, or use comparable nuclear dye, and then wash 3 times with PBS.
      CAUTION: DAPI is toxic and mutagenic. Wear proper personal protective equipment and dispose the toxic substance as instructed by institutional environmental health and safety procedures.
   7. Place PBS or PBS + 0.1% sodium azide (CAUTION) back onto the stained cells. Protocol can be stopped here and cells stored at 4 ˚C for several days, or proceed to image acquisition in section 5.
      ​CAUTION: Sodium azide is toxic. Wear proper personal protective equipment and dispose the toxic substances as instructed by institutional environmental health and safety procedures.
2. Analyze induction of base lesions or DNA adducts by IF staining.
   1. Fix and permeabilize cells in ice-cold methanol (CAUTION) for 20 min at -20 °C.
      CAUTION: Methanol is toxic and flammable. Wear proper personal protective equipment and dispose the toxic substance as instructed by institutional environmental health and safety procedures.
   2. Aspirate the methanol and allow the sample to dry completely for 15 min. Protocol can be stopped here and cells stored at -20 ˚C dry for up to 3 days.
   3. Rehydrate the cells in PBS for 15 min.
   4. Denature DNA using 2 N HCl (CAUTION) for 45 min at RT and wash 3 times with PBS.
      CAUTION: HCl is corrosive. Wear proper personal protective equipment and dispose the corrosive substance as instructed by institutional environmental health and safety procedures.
   5. Neutralize in 50 mM Tris-HCl pH 8.8 for 5 min followed by 3 washes with PBS.
   6. Incubate cells in blocking buffer made with 5% normal goat serum and 0.1% Triton X-100 in PBS for 1 h at RT.
   7. Incubate with primary antibodies for 8-oxo-2´-deoxyguanosine (8-oxodG, 1:400) or cyclobutane pyrimidine dimer (CPD, 1:1,000) in the goat serum blocking buffer for 1 h at RT, and then wash 3 times with PBS.
   8. Incubate cells with Alexa 488 goat anti-mouse at a 1:2000 ratio in the goat serum blocking buffer for 1 h at RT, and wash 3 times with PBS.
   9. Stain nuclear DNA using 1:5000 DAPI (10 mg/mL) in PBS for 5 min, and wash 3 times with PBS.
   10. If using chambered coverglass, place PBS or PBS + 0.1% sodium azide back onto the stained cells. Protocol can be stopped here and cells stored at 4 ˚C for several days, or proceed to image acquisition in section 5.
   11. Alternatively, mount cells in preferred mounting medium, if an additional coverglass can be placed on top of the culture vessel. Once cured, mounted cells can be stored at 4 ˚C for long periods of time.

5. Image acquisition for immunofluorescence experiments

1. Clean the coverglass bottom of the culture vessel with ethanol and place it back on the confocal microscope stage. Ensure that the orientation and position of the vessel on the stage corresponds to the orientation and position recorded when damage was induced. Secure the culture vessel to ensure optimal registration and imaging.
2. Locate previously imaged fields using the registration technique selected (described in 3.2).
   1. For manual registration using a coverglass with an etched grid, bring the grid into focus and find identifying marks. Then use recorded XY coordinates to locate damaged cells.
   2. For image-based automated registration, locate the structural feature used as a reference in step 3.2.1, and then align the current live microscope view as closely to the recorded image as possible. Once aligned, measure the current XY microscope stage location and compare it to the XY location of the reference image. The linear distance between the two locations defines the X and Y offset. Apply this offset to each recorded XY location to identify the image field(s) containing the irradiated cell. This offset function may be automated in the microscope control software or performed manually.
   3. If no appropriate registration points could be identified, manually locate cells by scanning the slide and locating the previously identified cell features.
3. Acquire images using appropriate microscope settings to collect all stained targets, including a brightfield image (setting in section 2.3 and focusing in section 3.3). In presented experiments, multichannel images were collected using the following excitation laser lines and fluorophores: 405 nm (DAPI), 488 nm (Alexa 488 and transmitted DIC brightfield), and 561 nm (Alexa 546). All lasers pass through a single acousto-optical tunable filter controlling the transmission of both laser wavelength and power.

6. Live cell image analysis of the recruitment of fluorescent proteins to micro-irradiated sites

1. Open acquired images in an image analysis application (i.e., NIS Elements or ImageJ). If necessary, combine the pre-irradiation image with timelapse images to generate a single image sequence of pre- and post- damage induction. Users can show recruitment by measuring changes in fluorescent intensity over the irradiated area relative to the fluorescence intensity measured across the whole nucleus (see Representative Results Figure 1B).
2. For each cell to be measured, first generate a reference ROI that represents the nucleus. Use a thresholding algorithm on the fluorescent signal that contains the pixels making up the nucleus, and then convert this area into a ROI. If necessary, the ROI can be manually drawn in using the brightfield as a reference. Adjust the ROI over the time course to ensure the nuclear area is accurately covered by the ROI, and report the mean fluorescence intensity of the fluorescent signal within this ROI for each frame.
3. For each cell to be measured create a 6×6 pixel ROI and place it over the damage ROI for each frame of the time course. This larger ROI is now the damage ROI for analysis. Report the mean ROI fluorescence intensity for each frame.
   NOTE: Most commercial image analysis software contains modules for 2D object tracking, and there are a number of user-developed macros for ImageJ or FIJI that facilitate faster workflow and higher throughput (see https://imagej.nih.gov/ij/plugins/).
4. Normalize the mean damage ROI fluorescence intensity to that of a corresponding reference ROI in each frame of the timelapse. Here, the mean nuclear fluorescence intensity is used as the reference ROI (see Representative Results Figure 1). Normalization can be performed by subtracting the mean reference ROI fluorescence intensity from the mean damage ROI fluorescence intensity, or by dividing the mean damage ROI fluorescence intensity by the mean reference ROI fluorescence intensity.
   NOTE: Normalization by division can yield unpredictable results, if used in situations with very low reference intensity values.
5. Repeat for all damaged cells, as well as for at least two control, undamaged cells. Control cell results can be used for further normalization, if required.
6. Graph normalized intensity values over time to show changes in recruitment dynamics as a function of experimental treatment.

7. Image analysis of protein recruitment detected by immunofluorescence

1. Open pre- and post-damage images in an image analysis application. The pre-damage images contain the damage ROI(s) used for micro-irradiation. Copy the ROI(s) and paste into the post-damage images to identify targeted cells.
2. Generate a 6×6 pixel ROI for analysis of protein recruitment and place this ROI at the location of the damage ROI. This larger ROI is now the damage ROI for analysis. Report the mean damage ROI fluorescence intensity.
3. Normalize the mean damage ROI fluorescence intensity to that of a corresponding reference ROI. Here, the mean nuclear fluorescence intensity of the damaged cell is used as the reference ROI (see Representative Results Figure 1). Generate the reference ROI by using a thresholding algorithm on the DAPI signal to define the nucleus, and then convert this area to a ROI. Report the mean reference ROI fluorescence intensity. Normalization can be performed by subtracting the mean reference ROI fluorescence intensity from the mean damage ROI fluorescence intensity, or by dividing the mean damage ROI fluorescence intensity by the mean reference ROI fluorescence intensity.
   NOTE: Normalization by division can yield unpredictable results, if used in situations with very low reference intensity values.
4. Repeat for all damaged cells, and perform the same analysis on at least two control cells, as described above.
5. Graph normalized intensity values for each measured micro-irradiation event against time to show changes in protein recruitment or type of DNA damage as a function of experimental treatment.