Name: Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord (Video) | JoVE Journal
Uploaded: 2015-02-22T16:00:00.000+00:00
Duration: 10 min 44 s
Description: Learn two-photon imaging of neural precursor cell dynamics in the mouse spinal cord using ex vivo preparation. This method enables real-time visualization of cellular interactions for neuroscience research.

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5 minutes

This work was supported in part by National Institutes of Health (NIH) Grants R01 GM-41514 (to M.D.C.), R39 GM-048071 (to I.P.), and R01 NS-074987 (to T.E.L.) and the National Multiple Sclerosis Society (NMSS) Collaborative Center Research Award CA1058-A-8 (to C.M.W., T.E.L. and M.D.C.), NMSS Grant RG4925, NIH Training Grant T32-AI-060573 (to M.L.G.), NMSS Postdoctoral Fellowship FG 1960-A-1 (to J.G.W.), and funding from the George E. Hewitt Foundation for Medical Research (M.P.M.).

NOTE: Ethics Statement: The protocol for animal handling was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California, Irvine, protocol #2010-2943.
1. Removal of Spinal Cord
<ol>
	<li>Place paper towels wetted with ~100% liquid isoflurane, USP in euthanizing chamber and place dry paper towels on top. Place mouse in chamber on top of dry paper towels so mouse is not touching the isoflurane and make sure the chamber is covered. Wait at least one minute after cessation of breathing to ensure that the mouse is euthanized.</li>
	<li>Perform a spinal transection of the neck to ensure the mouse is euthanized. Do not perform a cervical dislocation as this could damage the spinal cord.</li>
	<li>Spray the mouse with 70% ethanol to wet the hair.</li>
	<li>Use sharp Fine scissors to remove the hair and skin from the back of the mouse to expose the spinal column from approximately cervical lamina 1 (C1) to sacral lamina 4 (S4).</li>
	<li>Using a scalpel with a #10 blade, make incisions to the left and right of the spinal column to separate it from muscle and fat. This will make removal of the spinal cord much easier.</li>
	<li>optional: a curved Luer rongeurs can be used to scoop away additional flesh.</li>
	<li>While holding the top of the spinal vertebrae with serrated Graefe forceps, insert titanium curved Vannas scissors with the curved side up into the exposed spinal column at C1, being careful not to touch the spinal cord.</li>
	<li>Slide the titanium curved Vannas scissors all the way to the right and make one small cut. A small crunch sound should be heard and felt as the scissors cut the vertebrae. Repeat this cut on the far left side of the cord. Be careful not to go too quickly or make too large of a cut as this will risk damaging the cord with the scissors.</li>
	<li>The single lamina should be able to be lifted with the forceps once the sides of the vertebrae are cut. Repeat this step for each lamina until S4, continuing to hold and pull back the spinal cord vertebrae. 
	NOTE: the vertebrae above the transplant site will likely come off once it is cut down to the transplant site. Continue the procedure starting just below the transplant site.</li>
	<li>optional: lay the vertebrae down to see where the transplant site is and measure and cut ~20 mm rostral and caudal to the transplant site. 
	NOTE: When transplanting NPCs more will not be needed as the transplanted NPCs generally do not migrate farther than 15 mm. The amount of spinal cord needed will be dependent on experiment and migration characteristics of cell type transplanted. Making rostral and caudal cuts that are equidistant to the transplant site will enable the transplant site to be located more easily during imaging.</li>
	<li>Using a scalpel with a #11 blade carefully cut the ganglia on the right and left of the ventral side of the spinal cord starting at the rostral end. This will enable the spinal cord to be removed from the ventral vertebrae more easily. 
	NOTE: Done quickly (steps 1.7-1.11), the spinal cord will not dry out. However, 1x PBS can be administered to the cord to keep it moist.</li>
	<li>Invert the mouse and hold the mouse up so the spinal cord is facing down. Carefully peel out the spinal cord using closed serrated Graefe forceps. Without nicking the spinal cord, carefully cut any remaining ganglia to allow the spinal cord to be lifted out in a single intact piece.</li>
	<li>Make sure the spinal cord is in RPMI-1640 and place it on ice for transport to the 2P microscope.</li>
</ol>
2. Preparation of Spinal Cord for Imaging
<ol>
	<li>Embedding spinal cord in agarose
	<ol>
		<li>Keep the isolated spinal cord in chilled RPMI-1640 on ice prior to imaging.</li>
		<li>Weigh out agarose (low gelling temperature) and prepare a 5% solution in 5 ml of 1x PBS. Microwave the 5% agarose solution for 15 sec to dissolve the agarose and let the solution cool to 37 &#176;C.</li>
		<li>Prepare the spinal cord by placing it on a sheet of Parafilm with the ventral side facing up.</li>
		<li>Pipet approximately 5 ml of 5% agarose solution over the ventral side of the spinal cord. Let the agarose solidify by cooling to room temperature. Full solidification takes approximately 5 min.</li>
		<li>Apply a light coat of tissue adhesive to a 22 mm square cover slip.</li>
		<li>Invert the embedded spinal cord, placing the ventral side on Parafilm. The dorsal side will now be facing up. Adhere the cover slip to the dorsal side, and submerge the agarose embedded spinal cord/cover slip preparation in RMPI to solidify the adhesive. Remove excess solidified agarose using a razor blade.</li>
	</ol>
	</li>
	<li>Mounting the spinal cord on the microscope stage
	<ol>
		<li>Apply petroleum jelly to the bottom of the cover slip. This will stabilize the preparation during perfusion.</li>
		<li>Place the spinal cord preparation in an imaging well on the microscope stage with the ventral side facing up toward the 25X dipping objective. The custom-built imaging well is approximately 20 mm deep, 50 mm long, and 50 mm across.</li>
		<li>Image while superfusing the preparation with warmed (37 &#176;C), oxygenated (95:5 oxygen:carbon dioxide-carbogen) RPMI-1640 medium without serum.
		<ol>
			<li>Pre-warm media to 37 &#176;C in the water bath and connect it to the tubing pump by inserting the tubing into the media bottle.</li>
			<li>Retain the media temperature at 37 &#176;C a heater device at the connection of the tubing to the chamber. Superfuse media through well at 3 ml/min using tubing connected to the tubing pump (Figure 1B).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. 2P Imaging of Ventral Spinal Cord
<ol>
	<li>Microscope Setup
	<ol>
		<li>Acquire images from Thy1-eYFP spinal cords using a Chameleon Ultra Ti: Sapphire laser tuned to 900 nm. Attenuate laser power at the specimen to &#60;5% to ensure minimal phototoxicity16. Maintain temperature by perfusing oxygen-perfused RPMI-1640 at a constant 37 &#176;C using a single inline solution heater to ensure stable imaging, and to prevent tissue drift and cellular damage.</li>
		<li>To separate eGFP and eYFP fluorescent signal, place a 520 nm single-edge dichroic and a 560 nm single-edge dichroic beam splitter in series to separate 2P emission into three channels, purposely splitting the green emission to enhance visibility. 
		NOTE: These channel are referred to as blue-green (emission &#60;520 nm), green-yellow (520&#8211;560 nm), and red (emission &#62;560 nm). Photomultiplier tubes detect emitted light in each channel.</li>
	</ol>
	</li>
	<li>Locating imaging areas of interest
	<ol>
		<li>Using the eyepiece and a bright field light source, focus the dipping objective at the ventral edge of the spinal cord to set a reference point.</li>
		<li>Make sure ambient light source is off and switch to 2P excitation by opening the laser shutter.</li>
		<li>If available, when searching tissue for areas of interest, use a lower resolution setting, higher volume without digital zoom, and higher scan rate than when acquiring final images. Typical settings with this system when searching tissue for a region of interest are: resolution: 256 pixels; volume: x = 600 &#956;m, y = 600 &#956;m, z = 0-300 &#956;m.</li>
		<li>Observe the eYFP axons near the ventral edge of the spinal cord. Second harmonic signal from collagen (blue) will be brightest at the spinal cord tissue edge. Locate the eYFP axons just dorsal to the second harmonic signal from the collagen and eYFP signal is visualized in the green-yellow channel.</li>
		<li>Locate the transplant site in the longitudinal center of the spinal cord preparation14. For mice 1 day following eGFP-NPC transplantation, locate the transplant site by focusing the beam path in the z plane deep into the tissue. The transplant site will vary slightly between animals but is generally located ~200 &#956;m from the ventral edge. Observe the clusters of eGFP-NPCs in the white matter tracts, closer to the ventral and lateral edges in mice after day 1 post-transplantation.</li>
	</ol>
	</li>
	<li>Acquiring final images
	<ol>
		<li>Acquire image resolution of 512 pixels; volume: x = 270 &#956;m, y =212 &#956;m, and z = 100 &#956;m using Slidebook 6 software. Compile z-stacks by acquiring sequential focal planes in 2.5 &#956;m increments.</li>
		<li>Perform a bidirectional scan with proper interlace offset to ensure rapid quantification of any migrating objects in the spinal cord. Ensure that the ideal acquisition frame rate to determine cellular velocity within the spinal cord is approximately 1 frame/sec. 
		NOTE: Imaging settings can be changed to individual user preferences, such as acquisition frame rate, imaging resolution and imaging volumes. Larger imaging volumes will generally take longer to acquire at a given resolution.</li>
		<li>Analyze, crop, smooth, and pseudocolor Slidebook image files with image analysis software such as Bitplane Imaris 7.7. Produce final time-lapse videos by combing consecutive imaging volumes using an automated process in Slidebook and Imaris.</li>
	</ol>
	</li>
</ol>

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The authors have no competing interests.

Real-time 2P imaging of intact tissue is required to investigate NPC kinetics and interactions following transplantation into the demyelinated mouse spinal cord. Intravital 2P imaging is commonly used to determine cellular dynamics on the dorsal side of the spinal cord in living mice, and has been used to study dorsal demyelination in demyelinating disease17-19. However, because transplanted NPCs migrate to the ventral white matter, which lies too deep to image in situ using 2P microscopy, an ex vivo ...

Mouse models of demyelination, including experimental autoimmune encephalomyelitis (EAE) and intracranial infection with neuroadapted mouse hepatitis virus (MHV), are excellent tools to study molecular pathways and cellular interactions associated with disease. They have led to and supported the effectiveness of FDA approved pharmaceutical therapies, mainly targeting cessation of autoimmunity and inflammation1. However, once endogenous remyelination has failed, the currently approved therapies do not effectively repair demyelinated lesions in the central nervous system. Therefore, repair-focused therapies at this stage of disease are critical for the alleviation of chronic symptoms and improvement of quality of life. Recently, neural precursor cells (NPCs) have come to the forefront as a potential regenerative therapeutic modality to target areas of inflammation and demyelination. Several studies have highlighted the ability of NPCs to induce endogenous remyelination and participate directly in remyelination2-8. Because NPCs are involved in direct remyelination, it is imperative to understand their kinetics and interactions with endogenous cells following transplantation. After transplantation, NPCs migrate ventrally to areas of white matter damage, then rostrally and caudally relative to the transplant site5,9. The kinetics of migration differ in response to environmental cues; NPCs transplanted into a non-damaged spinal cord have greater velocities than NPCs transplanted into a damaged spinal cord6. After a migratory period, transferred NPCs proliferate extensively, at a higher rate in a damaged spinal cord relative to an intact spinal cord6. Finally, the majority of NPCs differentiate into oligodendrocytes and initiate direct remyelination4,6,9.
The demyelinated lesion is complex and can include a diverse population of cells at various stages of activation. For example, an active multiple sclerosis (MS) lesion may include a significant population of activated T cells, M1 microglia and M1 macrophages, but a chronic silent MS lesion may be comprised primarily of reactive astrocytes with few inflammatory cells10-13. Because of the diversity of effector cells, two-photon (2P) imaging in mouse models of demyelination is an extremely useful tool to help understand local cellular interactions within the lesion. In MS and many extensively used MS research models, the majority of lesions are located on the ventral side of the spinal cord, a region inaccessible to intravital 2P imaging due to lesion depth and the high lipid content of the spinal cord. To circumvent these issues and study cell-cell interactions within lesions along the ventral spinal cord we have developed a simple ex vivo 2P imaging preparation6.
This study follows up a previous methods publication, which showed the procedure for transplanting enhanced green fluorescent protein (eGFP)-expressing NPCs into the spinal cord of mice following JHMV strain of MHV-induced demyelination14. Five week old mice are infected with JHMV and transplanted with eGFP-NPCs at thoracic level 9 on day 14 post-infection. The protocol presented here provides detailed steps on how to extract the spinal cord, make an ex vivo agarose preparation, and image transplanted eGFP-NPC interactions with enhanced yellow fluorescent protein (eYFP)-expressing axons. Mice expressing eYFP under the neuronal-specific Thy1 promoter were used in this procedure15. Only some of the axons express eYFP, making it useful for imaging individual axons. Here we show spinal cords removed at 7 days post-transplant; however, spinal cords can be extracted at any time point following transplantation. While we show interactions of NPCs with damaged axons, our protocol can be used in combination with genetic fluorescent markers of other cell types to investigate a multitude of cellular interactions occurring throughout the mouse spinal cord.

<table><tbody><tr><td>Isoflurane, USP</td><td>Piramal Critical Care, Inc</td><td>N/A</td><td></td></tr><tr><td>Fine scissors</td><td>Fine Science Tools</td><td>14060-09</td><td>sharp</td></tr><tr><td>scalpel blade #10</td><td>Fine Science Tools</td><td>10010-00</td><td></td></tr><tr><td>scalpel handle</td><td>Fine Science Tools</td><td>10003-12</td><td></td></tr><tr><td>Luer rongeurs</td><td>Fine Science Tools</td><td>16001-15</td><td></td></tr><tr><td>Graefe forceps</td><td>Fine Science Tools</td><td>11052-10</td><td></td></tr><tr><td>Vannas scissors</td><td>Fine Science Tools</td><td>15615-08</td><td></td></tr><tr><td>scalpel blade #11</td><td>Fine Science Tools</td><td>10011-00</td><td></td></tr><tr><td>RPMI-1640</td><td>Gibco</td><td>12-115F</td><td></td></tr><tr><td>agarose, low gelling temperature</td><td>Sigma</td><td>A9414-25G</td><td></td></tr><tr><td>Parafilm</td><td>Fisher Scientific</td><td>13-374-12</td><td></td></tr><tr><td>Vetbond (tissue adhesive)</td><td>3M</td><td>1469SB</td><td></td></tr><tr><td>22 mm square cover slip</td><td>Fisher Scientific</td><td>12-547</td><td></td></tr><tr><td>25X dipping objective, 1.1 NA</td><td>Nikon</td><td>CFI Apo LWD 25XW</td><td></td></tr><tr><td>Single inline solution heater</td><td>Warner Instruments</td><td>64-0102</td><td></td></tr><tr><td>520 nm single-edge dichroic beam splitter</td><td>Semrock</td><td>FF520-Di02-25x36</td><td>Brightline</td></tr><tr><td>560 nm single-edge dichroic beam splitter</td><td>Semrock</td><td>FF560-FDi01-25x36</td><td>Brightline</td></tr><tr><td>photomultiplier tubes</td><td>Hamamatsu</td><td>R928</td><td></td></tr><tr><td>C/L variable-speed tubing pump</td><td>Masterflex</td><td>77122-22</td><td></td></tr><tr><td>digital thermometer</td><td>Comar Instruments</td><td>3501</td><td></td></tr><tr><td>Chameleon Ultra Ti:Sapphire laser&amp;nbsp;</td><td>Coherent</td><td>N/A</td><td></td></tr><tr><td>Slidebook 6 software</td><td>3i</td><td>N/A</td><td></td></tr><tr><td>Imaris 7.7 software</td><td>Bitplane</td><td>N/A</td><td></td></tr></tbody></table>

two-photon imaging of cellular dynamics in the mouse spinal cord

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

While the explanted spinal cord imaging protocol can be used to visualize any fluorescence within the spinal cord, our representative results demonstrate eGFP-NPC interactions with eYFP-axons. First, we show the embedded ventral spinal cord preparation in Figure 1A. Next, we show the 2P microscope setup and key components in Figure 1B. Figure 2 demonstrates eGFP and eYFP fluorescence in a single z-stack within the ventral spinal cord. Acquisition of consecutive z-stacks ...

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

The overall goal of the following experiment is to image fluorescently labeled transplanted, neural precursor cell or NPC interactions with fluorescent axons in the mouse spinal cord in real time. This is achieved by carefully removing and embedding the spinal cord from mice transplanted with NPCs into 5%aros and attaching it to a cover slip. The orientation of the spinal cord can be altered to view fluorescence in any region of the spinal cord.In the second step, the spinal cord preparation is placed on the two photon imaging stage for imaging of viable NPCs while being super fused with warm oxygenated RPMI media for up to 10 hours. An appropriate single edge diic beam splitter is used to separate the YFP and GFP emissions. Ultimately, two photon imaging allows visualization of the interactions of the GFP expressing NPCs with the YFP expressing axons in the damaged spinal cord in real time.This method can help answer key questions in the field of neurogeneration, such as what are the molecular and cellular signaling cues necessary for transplanted NPC remyelination of damaged axons, and what are the kinetics of these remyelination events? The main advantage of this technique over existing methods such as intertidal two photon imaging, which can be only used to image the dorsal spinal cord, is that this method allows imaging of transplanted NPCs that have migrated deep within the ventral region of the spinal cord. After confirming euthanasia by spinal transection, wet the mouse with 70%ethanol, then use sharp fine scissors to remove the hair and skin from the back of the animal, exposing the spinal column from approximately the cervical lamina one or C one to the sacral lamina four or S four.Next, using a scalpel with a number 10 blade, make incisions to the left and right of the spinal column to separate it from the surrounding muscle and fat tissue also cut away excess tissue around and on top of the spinal column. This will allow for easier removal of the vertebrae. Then while holding the top of the spinal vertebrae with serrated gray forceps, insert titanium curved van scissors with the curved side up into the exposed spinal column at C one, taking care not to touch the spinal cord.Slide the scissors all the way to the right and make one small cut. A small crunching sound should be heard and felt as the scissors cut the vertebrae. Repeat this cut on the far left side of the cord, taking care not to work too quickly or to make too large of a cut, as this will risk damaging the cord.Once the sides of the vertebrae are cut, the single lamini should be able to be lifted with the forceps. Repeat the left and right cuts for each lamina until S four is reached. Continuing to hold and pull back the spinal cord vertebrae.The vertebrae above the transplant might come off once his spine is cut down to the transplant site. Once the vertebrae are cut down to S four, lay the vertebrae down to help visualize where the transplant site is and cut approximately 20 millimeters rostral and coddle to the transplant site. Now use a scalpel with a number 11 blade to carefully cut the ganglia on the right and left of the ventral side of the spinal cord, starting at the rostral end.Then invert and hold the animal up so that the spinal cord is facing down and use closed serrated grave forceps to carefully peel out the spinal cord from the coddle end. Then without nicking the spinal cord, carefully cut any of the remaining ganglia to allow the spinal cord to be lifted out in a single intact piece, and place the cord in medium on ice to prepare the spinal cord for imaging. Transfer the nervous tissue to a sheet of param with the ventral side facing up.Then pipette approximately five milliliters of freshly prepared 37 degrees Celsius, 5%agro solution over the cord, and let it solidify at room temperature. After about five minutes, apply a light coat of tissue adhesive to a 22 millimeter square cover slip and invert the embedded spinal cord ventral side down onto a new piece of perfil. Adhere the cover slip to the exposed dorsal side and submerge the entire preparation in fresh medium to solidify the adhesive.Then use a razor blade to remove the excess solidified aga rose. Next, insert the tubing connected to the tubing pump into a media bottle, kept in a water bath to super fuse 37 degrees Celsius. Pretty warmed media through a custom built imaging well on the microscope stage at three milliliters per minute.Then place the spinal cord preparation into the well with the ventral side facing up toward the 25 x dipping objective. To image the ventral spinal cord by two photon microscopy, use an ultra titanium sapphire laser tuned to 900 nanometers with the laser power attenuated at the specimen to less than 5%to ensure minimal phototoxicity. Then using the eyepiece and a bright field light source focus the dipping objective at the ventral edge of the spinal cord.To set a reference point, make sure the ambient light source is off. Then open the laser shutter to switch the two photonic citation if available. When searching the tissue for areas of interest, use a lower resolution setting a higher volume without digital zoom and a higher scan rate than when acquiring the final images.To find the cells, it is best to focus on the sample under bright field illumination at the tissue edge, at the transplant site first, and then to switch to fluorescence. A large X and Y field with a low resolution can then be used to enable the quick viewing of a large area as the focus is move deeper into the tissue. Observe the EYFP axons near the ventral edge of the spinal cord.The second harmonic signal from the collagen observed here in blue will be the brightest at the spinal cord tissue edge. Now, use the appropriate image capture software to acquire the final higher resolution images in sequential focal planes in 2.5 micron increments to compile Z stacks. In this representation, GFP NPCs are observed as blue and damaged YFP axons are observed as yellow pseudo coloring can be performed later using editing software then perform a bi-directional scan with the proper interlay offset to ensure the rapid quantification of any migrating objects in the spinal cord, ensuring that the ideal acquisition frame rate to determine the cellular velocity within the spinal cord is set to approximately 1.7 frames per second.Finally, analyze crop smooth and pseudo color the image files with the appropriate software, and use the automated processes to comb the consecutive imaging volumes for producing the final time lapse videos. While this explanted spinal cord imaging protocol can be used to visualize any fluorescence within the spinal cord, these images demonstrate representative EGFP neural precursor cell interactions with EYFP axons in a single Z stack within the ventral spinal cord. The acquisition of consecutive Zacks can then be compiled to produce time lapse videos for analyzing the real-time cellular dynamics within the intact tissue.Indeed use of a 520 nanometer single edge dichroic and a 560 nanometer single edge dichroic beam splitter can separate the EGFP and EYFP signals, allowing pseudo coloring of the individual channels in green and yellow with the imaging software as demonstrated here, While attempting this procedure, it's important to remember to ensure the proper removal of the spinal cord from the mouse without excessive stretching, compressing, or accidental cutting of the nervous tissue. With this Procedure, any combination of fluorescently labeled cells or axons can be imaged in real time to answer additional questions, including what are the kinetics of migration or remyelination under various drug treatments or conditions.

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Research

JoVE Journal

Neuroscience

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In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

Source: Maruoka, H., et. al. Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice. J. Vis. Exp. (2022)     This video shows a two-photon microscopy procedure for imaging neuronal activity and microglial dynamics in an awake mouse during visual stimulation, highlighting calcium transients and cellular interactions.

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in an Awake Mouse

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Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

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A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

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An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

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Laminectomy and Spinal Cord Window Implantation in the Mouse

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Longitudinal Two-Photon Imaging of Dorsal Hippocampal CA1 in Live Mice

Stimulated Raman scattering (SRS) microscopy allows label-free imaging of biomolecules based on their intrinsic vibration of specific chemical bonds. In this protocol, the instrumental setup of an integrated SRS and two-photon fluorescence microscope is described to visualize cellular structures in the spinal cord of live mice.

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice.

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Learn two-photon imaging of neural precursor cell dynamics in the mouse spinal cord using ex vivo preparation. This method enables real-time visualization of cellular interactions for neuroscience research.

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

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