Optical methods, such as optogenetics can provide the precise modulation of cellular molecular signals. Our protocol demonstrated an oral optical manipulation of cellular signal moleculars by femtosecond laser stimulation. By coupling our femtosecond laser into a confocal microscope, our technique can provide single-cell or subcellular-level operation by femtosecond laser stimulation and the real-time, molecular dynamic monitoring by confocal microscopy.
This method can be applied to any fluorescent microscopy systems by coupling a femtosecond laser into the system in the proper manner. For anyone who is trying our method for the first time, please follow the separate instructions of the laser facility or seek guidance from professionals when operating a lab femtosecond laser source. To begin, turn on the femtosecond laser and the confocal microscope.
Use reflective mirrors to direct the femtosecond laser beam through a mechanical shutter. Next, set a relay telescope consisting of a pair of lenses to expand the transmission beam width. This should be consistent with the back aperture of the objective.
Now, steer the reflective mirrors to align the expanded beam into the microscope. Adjust the two reflective mirrors, pointed out here, to tune the focus of the femtosecond laser to the center of the field of view. Finally, measure and tune the lasers as described in the accompanying text protocol.
Culture Hela cells using standard techniques. When ready to perform the experiment, transfect the cells with ERK2-GFP as described in the accompanying text protocol. Next, turn on the laser scanning confocal microscope and the femtosecond laser.
Ensure that the laser's shutter is closed. Then, open the microscope software. Set the excitation laser at 488 nanometers, and set its power level at 0.1 milliwatts.
Set the imaging size as 512 by 512 pixels, and the interval time of each pixel as 2.4 microseconds. Then, set the interval time between two frames at six seconds to minimize photo bleaching and photo damage to the cells. Also, set the total imaging frames at around 300 frames to provide about 30 minutes of continuous microscopy in an individual experiment.
Take the dish containing the Hela cells transfected with ERK2-GFP from the incubator, and put the dish on the microscope stage. In the software, start fast scanning mode, and tune the objective to acquire clear fluorescent images of the cells. Then, stop fast scanning and switch to bright-field imaging mode using the CCD camera.
Open the shutter and adjust the distance between the two lenses of the relay telescope to ensure that the diameter of the femtosecond laser focus is about two microns. Then, close the shutter to complete the adjustment. Set the power of the femtosecond laser at 15 to 40 milliwatts for an 810 nanometer laser, or a 20 to 60 milliwatts for a 1040 nanometer laser.
Then, set the shutter opening time at 05 to 0.2 seconds. Use fast scanning mode to select a target cell that is strongly expressing ERK2-GFP. Once found, move the stage in order to localize the cytosol area of the selected target cell so that it is at the center of the field of view when under bright-field imaging.
Once centered, click on the start button to begin continuous imaging. Open the shutter at any predefined time slot to deliver the femtosecond laser stimulation into the target cell. When the imaging process is complete, save the imaging data for further analysis.
Set the power of the femtosecond laser at 15 to 40 milliwatts and 810 nanometers and 20 to 60 milliwatts and 1040 nanometers at the specimen. Then, take the dish containing cells transfected with ERK2-GFP from the incubator and put it on the microscope stage. Use fast scanning mode to select the target cell well expressing ERK2-GFP.
Then, set the confocal imaging process and define a special scanning frame as the stimulation frame in the imaging process. Define the parameter of the stimulation frame. Set a scanning region of two by two to three by three square microns at the cytosol area close to the nucleus in the target cell.
Set the total scanning time at 0.1 to 0.2 seconds. Set the interval time between two frames at six seconds and set the total imaging frames at around 300 frames to provide about 30 minutes of continuous microscopy. Save the imaging data for further data analysis.
Synchronize the shutter of the femtosecond laser with that of the confocal scanning according to the predefined photo stimulation area which is only open when the laser scanning drops in the stimulation frame. Click the start button to start the continuous microscopy imaging process. To observe mitochondrial morphological dynamics, transfect Hela cells with Mito-GFP to fluorescently indicate the mitochondria, or a base with mito-cpYFP to observe mitoflashes.
Following transfection, turn on the femtosecond laser and ensure the shutter is closed. Then, turn on the laser scanning confocal microscope. Set the excitation laser at 488 nanometers.
Then, set the power level of the 488 nanometer laser at 0.1 milliwatt. Additionally, set the imaging size to 512 by 512 pixels and the total imaging time of each frame to 2.2 seconds. Then, set the interval time between two frames at zero seconds and the total imaging frames as 200 frames to provide about 440 seconds of continuous microscopy in an individual experiment.
Take the dish containing cells transfected with mito GFP or mitochondrial-targeted circularly permuted yellow fluorescent protein from the incubator and put the dish on the microscope stage. Check the status of the femtosecond laser to ensure that the focus of the femtosecond laser is located in the center of the field of view. Then, set a reference arrow at the center of the fluorescent imaging window to indicate the position of the focus.
Next, set the power of the femtosecond laser at five to 30 miliwatts with the laser at 810 nanometers and 10 to 40 milliwatts for the laser at 1040 nanometers. Set the opening time of the shutter to 05 to 0.1 seconds. Use fast scanning mode to select the target cell that is well expressing mito GFP or the mitochondrial-targeted circularly permuted yellow fluorescent protein.
Select one mitochondrion randomly in the target cell as the experimental subject. Next, move the microscope stage in order to localize the target mitochondrial tubular structure at the center of the field of view by fast scanning mode as indicated by the reference arrow. Click the start button to start continuous imaging.
Finally, open the shutter at any predefined time to deliver the femtosecond laser stimulation into the target mitochondrial tubular structure. Wait until the imaging process is complete and then save the imaging data for further data analysis. Using the method presented in this protocol, ERK2 translocates into the nucleus after being treated with a short flash of the femtosecond laser.
ERK2-GFP fluorescence reaches the maximum after several minutes of femtosecond laser stimulation. The ERK2 molecules will be dephosphorylated after activating downstream substrates in the nucleus and then the ERK2 comes back to the cytoplasm indicated by a decreasing of nuclear GFP fluorescence. ERK2 can be activated multiple times by multiple photo stimulations.
Therefore, it is possible to manipulate the ERK2 signal pattern precisely by controlling interval time between multiple stimuli. In addition, ERK2 can occasionally be activated in adjacent cells around the stimulated cell. This observation indicates that some diffusible molecules may be released by the cell treated with the femtosecond laser to activate ERK2 in the adjacent cells.
Phosphorylation of ERK2 downstream protein eIF4E can be confirmed individualized by immunofluorescence microscopy. This result indicates that femtosecond laser stimulation can successfully activate the ERK signaling pathway. Mitochondrial oxidative flashes or mitoflashes are oxidative bursts in mitochondria that root from complex molecular dynamics.
Implementing this photo stimulation scheme resulted in a successful mitoflash excitation. This photo stimulation also shows varieties of mitochondrial molecular dynamics including fragmentation and restoration of mitochondrial morphology as well as oscillation of mitochondrial membrane potential. Similar to photo activated mitoflashes, these mitochondria events have different performance with different power intensities.
The most important thing is to make sure that the focus, size and the precision of the femtosecond laser is a property for the stimulation in cells. The near-infrared femtosecond laser may be hazardous to users. When following this protocol, wear proper protection and it prevents the laser from diffusing out from the optical table.
