The work was supported by National Natural Science Foundation of China (81571719 and 81661168014, 81673014, and 81870055), Shanghai Municipal Science and Technology Committee (18QA1402300 and 16XD1403100), and National Key R&#38;D Plan (2017YFA0104600).

CAUTION: The protocol presented below involves using NIR femtosecond laser and toxic chemicals. Please pay attention to all possible damages induced by experiment procedures. Please read safety data sheets of all relevant chemicals or other materials before use. Please follow the safety instructions of the laser facilities or consult professionals for guidance before operate laser source.
1. Experimental Preparation
<ol>
	<li>Setting up the photostimulation system</s...

<ol>
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We demonstrate a photostimulation strategy by combining a femtosecond laser with a laser scanning confocal microscope system. The photostimulation can directly work as a two-photon microscopy by defining Stim-B accordingly. We provide a detailed protocol for utilizing a short flash of femtosecond laser to trigger ERK signaling or mitoflashes in target cells. The different stimulation modes can be performed according to different experimental purposes and systems. Stim-A can be easily set up based on a confocal microscope...

The technology of controlling cellular signaling molecules is an important part of the development of life science. Traditionally, the most commonly-used method is biochemical treatment by drugs or biological materials1,2,3. Over the past decade, the invention of optogenetics opens a new era for cellular molecular signal modulation. Transfection with light sensitive proteins by gene engineering makes light become a powerful tool to modulate various protein activities in target cell. This technology has made encouraging progresses such as excitation and inhibition of neural signal, promoting gene expression, manipulating cellular signal patterns, leading different cell fates and pathological investigation4,5,6,7,8,9. However, light can only work by transfecting cells with optogenetic proteins. In current stage, there exist rare methods that enable light to control cellular molecules directly besides optogenetics.
Femtosecond laser has advanced biological researches by providing efficient multiphoton excitation while maintaining good biological safety. By deploying diverse photo processing strategies, it has realized numerous achievements such as multiphoton microscopy, microsurgeries and multiphoton optogenetic applications10,11,12,13,14,15,16. Recent investigations show that femtosecond laser stimulation has been demonstrated as a highly efficient optical method to directly induce molecular signaling events. It has been found that tightly-focused femtosecond laser irradiation on endoplasmic reticulum (ER) is able to deplete calcium in ER and activate calcium-release-activated calcium (CRAC) channels to form calcium signals in cells17. This photo-activated calcium signal can spread between multiple types of cells18,19,20. Furthermore, it also has the ability to activate cell signaling pathways such as nuclear factor of activated T cells (NFAT) and ERK signaling pathway21,22. By adjusting the intensity and localization of femtosecond laser exposure in cells, for example, focusing the laser on mitochondria, it can influence mitochondrial morphology and molecular events23,24,25. Specifically, bursts of mitochondrial ROS generation can be excited by photostimulation, which is remarked as fluorescent flashes in mitochondria (mitoflashes).
Hence, the photostimulation technology is of good potential to be widely applied in related biological research. It is also a good chance to extend femtosecond laser applications in controlling of cellular signaling molecules and functions besides microscopy. Here, we provide the technical details of photostimulation. The photostimulation is achieved by coupling a femtosecond laser to a confocal microscope to provide single target cell with a short flash photostimulation. It can initiate efficient and controllable ERK activation in the cell. If the photostimulation is located on the mitochondrial tubular structure, the mitochondrial membrane potential, morphology, ROS, and permeability transition pores, can all be controlled by the photostimulation. Based on this photostimulation scheme, we provide a detailed method of activating ERK signaling pathway and influencing multiple mitochondrial events in Hela cells. This protocol elucidates the process of delivering femtosecond laser stimulation into target cells.
The photostimulation system is established on a confocal microscope with a femtosecond laser coupling into it for simultaneous stimulation and continuous microscopy. The femtosecond laser (wavelength: 1,040 nm, repetition rate: 50 MHz, pulse width: 120 fs, maximum output average power: 1 W) is split into two beams before coupling. One is guided through a relay telescope consisting of a pair of lenses. It is then directly reflected into the back-aperture of an objective (60x, N.A. = 1.2, water immersion) to form a diffraction-limit focus (Stim-A). The other is reflected to the scanning optical path of confocal microscope to work as a two-photon scanning mode (Stim-B). Stim-A presents a fixed focus point in the center of field of view (FOV). Stim-B is a pre-designed partial confocal scanning area in the FOV. Stim-A and Stim-B are shown in Figure 1A. A CCD camera under the dichroic mirror (DM) provides bright-field imaging for monitoring the focus of femtosecond laser.
There are some crucial essentials for the following experiments. In this protocol, a femtosecond fiber laser source (1040 nm, 50 MHz, 120 fs) is used as an example. In practice, most commercial femtosecond oscillators can be used as long as the pulse width is shorter than 200 fs and the peak power density should be above the level of 1011 ~ 1012 W/cm2. For example, a Ti: Sapphire laser usually used for multiphoton microscopy is able to replace the femtosecond laser showed in Figure 1B. The laser power and some other photostimulation parameters need to be tuned because the optical parameters (pulse width, wavelength, and repetition rate) vary a lot in different femtosecond lasers that thus induce different multiphoton excitation efficiencies.
Along with femtosecond laser stimulation, the confocal microscopy provides continuous cell imaging to monitor molecular dynamics in real time in both Stim-A and Stim-B modes. Both photostimulation schemes (Stim-A and Stim-B) are controlled by a mechanical shutter with millisecond resolution (Figure 1).
In Stim-A mode, the position of laser focus is fixed in the center of FOV. A relay telescope is used to ensure the focus of femtosecond laser to be located on confocal imaging plane by tuning the distance between two lenses in the vertical direction (the laser propagation direction, vertical to the confocal imaging plane, as shown in Figure 1). By the bright-field imaging of CCD camera, the diameter of laser focus can be measured (~2 &#181;m, Figure 2B). The stimulation durations and exposure times are controlled by a shutter during the confocal imaging process.
In Stim-B mode, the stimulation area can be pre-assigned manually in the confocal imaging controlling software to any form like line, polygon, or circle. The shutter is synchronized with the confocal scanning process. It opens at the pre-designed time which is set through the confocal imaging controlling software. Then, the stimulation area is scanned by femtosecond laser as confocal microscopy. Thus, the sample is only stimulated by femtosecond laser when confocal scanning process enters a given imaging frame.
The photostimulation system can be established on both inverted and upright metallurgical microscopes according to the experiment subjects. In vitro cells cultured in Petri dishes are better to work with inverted microscopes. Animals, especially brains of live animals, are more suitable with upright microscopes. In this study, we take the inverted microscope as an example. It should be noted that the cover of Petri dish is not opened during the whole experiments.

<table>
	<tbody>
		<tr>
			<td>inverted microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>femtosecond laser</td>
			<td>Fianium</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubation system</td>
			<td>Olympus</td>
			<td>MIU-IBC</td>
			<td></td>
		</tr>
		<tr>
			<td>petri dish</td>
			<td>NEST</td>
			<td>801002</td>
			<td></td>
		</tr>
		<tr>
			<td>petri dish with imprinted grid</td>
			<td>Ibidi</td>
			<td>81148</td>
			<td></td>
		</tr>
		<tr>
			<td>ERK-GFP</td>
			<td>addgene</td>
			<td>37145</td>
			<td>A gift from Rony Seger&#39;s lab</td>
		</tr>
		<tr>
			<td>mt-cpYFP</td>
			<td></td>
			<td></td>
			<td>A gift from Heping Cheng&#39;s lab</td>
		</tr>
		<tr>
			<td>mito-GFP</td>
			<td>Invitrogen</td>
			<td>C10508</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylrhodamine (TMRM)</td>
			<td>Invitrogen</td>
			<td>T668</td>
			<td>Dilute in DMSO</td>
		</tr>
		<tr>
			<td>polyethylenimine (PEI)</td>
			<td>Sigma-Aldrich</td>
			<td>9002-98-6</td>
			<td>Dilute in PBS</td>
		</tr>
		<tr>
			<td>paraformaldehyde</td>
			<td>Solarbio</td>
			<td>P8430</td>
			<td>Dilute in PBS</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Solarbio</td>
			<td>T8200</td>
			<td>Dilute in PBS</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>9048-46-8</td>
			<td>Dilute in PBS</td>
		</tr>
		<tr>
			<td>Tween20</td>
			<td>Sigma-Aldrich</td>
			<td>9005-64-5</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-eIF4E antibody</td>
			<td>abcam</td>
			<td>ab76256</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Bax antibody</td>
			<td>abcam</td>
			<td>ab53154</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-cytochrome C antibody</td>
			<td>abcam</td>
			<td>ab90529</td>
			<td></td>
		</tr>
		<tr>
			<td>secondary antibody (anti-Rabbit IgG H&#38;L, Alexa Fluor 488)</td>
			<td>abcam</td>
			<td>ab150077</td>
			<td></td>
		</tr>
	</tbody>
</table>

photostimulation by femtosecond laser activates extracellular-signal-regulated kinase (erk) signaling or mitochondrial events in target cells

Direct control of cellular defined molecular events is important to life science. Recently, studies have demonstrated that femtosecond laser stimulation can simultaneously activate multiple cellular molecular signaling pathways. In this protocol, we show that through coupling femtosecond laser into a confocal microscope, cells can be stimulated precisely by the tightly-focused laser. Some molecular processes that can be simultaneously observed are subsequently activated. We present detailed protocols of the photostimulation to activate extracellular signal regulated kinase (ERK) signaling pathway in Hela cells. Mitochondrial flashes of reactive oxygen species (ROS) and other mitochondrial events can be also stimulated if focusing the femtosecond laser pulse on a certain mitochondrial tubular structure. This protocol includes pretreating cells before photostimulation, delivering the photostimulation by a femtosecond laser flash onto the target, and observing/identifying molecular changes afterwards. This protocol represents an all-optical tool for related biological researches.

The photostimulation can be performed simultaneously along with continuous confocal scanning microscopy. The photostimulation can start at any pre-defined time slot in the time-lapse confocal microscopy sequence. The confocal microscopy can monitor cellular molecules by fluorescent imaging. The molecular responses to photostimulation and other dynamics can be identified in this way. Theoretically, if ERK is activated, it will be phosphorylated move from the cytoplasm to cell nucleus<sup c...

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Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase ERK Signaling or Mitochondrial Events in Target Cells

Tightly-focused femtosecond laser can deliver precise stimulation to cells by being coupled into a confocal microscopy enabling the real-time observation and photostimulation. The photostimulation can activate cell molecular events including ERK signaling pathway and mitochondrial flashes of reactive oxygen species.

Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase (ERK) Signaling or Mitochondrial Events in Target Cells

Direct control of cellular defined molecular events is important to life science. Recently, studies have demonstrated that femtosecond laser stimulation ...

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.

photostimulation-femtosecond-laser-activates-extracellular-signal

Research

JoVE Journal

Biology

6.7K visualizações.  Shanghai Jiao Tong University. Métodos ópticos, como a optogenética, podem fornecer a modulação precisa de sinais moleculares celulares. Nosso protocolo demonstrou uma manipulação óptica oral de sinais moleculares de sinal celular por estimulação a laser femtosegundo. Ao acoplar nosso laser femtosegundo em um microscópio confocal, nossa técnica pode fornecer operação de nível único ou subcelular por estimulação a laser femtosegundo e o monitoramento dinâmico molecular em tempo real por microscopia confoc...