Name: photostimulation by femtosecond laser activates extracellular-signal-regulated kinase (erk) signaling or mitochondrial events in target cells
Uploaded: 2019-07-06T14:00:00
Description: Watch this Scientific Journal Video about Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase ERK Signaling or Mitochondrial Events in Target Cells at JoVE.com

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...

Watch this Scientific Journal Video about Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase ERK Signaling or Mitochondrial Events in Target Cells at JoVE.com

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 ...

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Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...

Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent ...