The authors would like to thank Neurotar Ltd. for technical assistance, CIMO Foundation and FGSN for financial support.

Procedures involving animal subjects have been approved by the National Animal Experiment Board, Finland.
1. Animal Preparation for Imaging
<ol>
	<li>Anesthetize a mouse by intraperitional (IP) injection of ketamine (0.08 mg per body weight) and xylazine (0.01 mg per body weight). Check the anesthesia with the rear toe pinch reflex.</li>
	<li>Immerse animal&#8217;s eyes in eye drops (Viscotears) to protect the eyes from dehydration.</li>
	<li>Put the mouse on a heating pad (Supertech) at 37&#160;&#176;C to prevent hypothermia.</li>
	<li>Clean the foot pad designated for imaging with 70% ethanol.</li>
	<li>Add a drop of water on the skin of the foot pad for the immersion coupling between skin and cover glass.</li>
	<li>Put plastic packaging material under the hind paw that is positioned under a metal ring with cover class.</li>
	<li>Adjust the thickness of the plastic packaging material to flatten the skin.</li>
	<li>Put a drop of water on the cover glass for immersion between cover glass and the objective.</li>
</ol>
2. Metal Fixator Ring Preparation
<ol>
	<li>For the stabilization of the skin utilize the metal fixator. We use Community Design-protected two-wing fixator with a metal ring (courtesy of Neurotar Ltd, Finland). Fill the syringe with superglue, put the small drop of superglue through the needle on the ring and gently spread the glue for uniform coverage of the ring surface. It is critical to put the minimal amount of glue that is needed only for uniform coverage, because excessive amount of glue may reduce the field of the view and obstruct subsequent imaging. 
	NOTE: Alternatively the combination of a metal bar (from beneath) and standard microscope glass slide (as a cover) could be used to flatten the skin and to ensure proper optical coupling of the assembly14. Two paper clips could be used to fix the paw between glass and metal bar surface (Figure 4).&#160;</li>
	<li>Cover the ring with microscope cover slide (5 mm diameter, Electron Microscopy Science).</li>
	<li>Screw on the ring fixator to the rigid metal bar that is montaged on the motorized microscope stage.</li>
</ol>
3. Imaging Procedure
<ol>
	<li>In case of using thy1-YFP-H mice, choose the blue part of fluorescence lamp spectra to visualize nerve fibers. Find the nerve of interest in epifluorescence mode and focus on it.</li>
	<li>Write down the coordinates of a spot for longitudinal imaging. Use a carpal pad as a reference point. During the following imaging sessions, detect first the carpal pad and then find the same upstream big nerve fibers and go back using the saved coordinates. It is essential to position the foot pad in the same direction perpendicular to the metal bar to keep the same system of reference.</li>
	<li>Turn on the two-photon mode. For the thy1-YFP-H mice, it is suitable to use the 950 nm wavelength of laser radiation to visualize the nerve fibers.</li>
	<li>Select laser wavelength and emission channels (from 450 to 480 nm for second harmonic generation detection, from 520 to 550 nm for imaging of YFP-labeled nerves and from 580 to 630 nm for detection of YFP fluorescence and autofluorescence of the hair), start with low laser power (3-5%) to prevent photodamage and bleaching. The typical voltage on the photomultiplier tubes is 600-700 V for this kind of imaging.</li>
	<li>Set the desired resolution (512 x 512 or 640 x 640 for fast events measurements, 800 x 800 or 1,024 x 1,024 for morphological imaging), axial step (1-3 &#181;m), thickness of the sample and the time intervals (1-10 min). The exposure time is in the order of 1 msec per one pixel.</li>
	<li>First mark the upper and lower boundaries of the imaging volume in the software.</li>
	<li>Acquire the reference image stack for the reference before the lesion. When needed, it is possible to record the baseline during several minutes.</li>
	<li>Repeatedly (at least every 10-15 min) check animal breathing rate and reflexes, when needed apply an additional amount of anesthetics.</li>
	<li>After imaging clean the pad with a tissue and put the mouse in a recovery box at 36 &#176;C until it is fully awake. 
	NOTE: Usually, the duration of one imaging session combined with laser induced lesion (see below) does not exceed 1 hr. We recommend verifying that the animal is deeply anaesthetized every 10-15 min; this should not interfere with imaging itself but should be considered while experimental procedure is planned. The duration of the effect of a single ketamine/xylazyne dose is around 30 to 40 min, thus we recommend to apply additional &#188; to &#189;&#160; dose after 25-30 min. 
	NOTE: It should be considered as well when the experimentation is planning that the frequency of imaging sessions for an individual animal should not exceed 1 session per 2 days to avoid adverse effects of repetitive anesthesia.</li>
</ol>
4. Laser Lesion
<ol>
	<li>After acquiring the reference image stack, use the bleaching protocol to make a micro lesion.</li>
	<li>Increase the laser power to 100%.</li>
	<li>Increase the exposure time to 100-1,000 msec per region of interest (100-1,000x&#160;more than in case of standard imaging).</li>
	<li>Outline the area for the lesion.</li>
	<li>Make the lesion by switching on the bleaching.</li>
	<li>Switch back to the regular imaging mode and continue with time-lapse recordings.</li>
</ol>
5. Data Processing and Analysis
<ol>
	<li>Proceed with unmixing using Spectral Unmixing plugin in ImageJ8 (Joachim Walter, <a href="http://rsbweb.nih.gov/ij/plugins/spectral-unmixing.html">http://rsbweb.nih.gov/ij/plugins/spectral-unmixing.html</a>).</li>
	<li>Open the image stack and split the channels.</li>
	<li>Find the area free of the nerves or hair for the background measurements. Put a region of interest in that area.</li>
	<li>Outline carefully the part of the hair where it is clearly distinguishable from the nerves.</li>
	<li>Outline the nerve in the part of the image where it is separate from the hair.</li>
	<li>Save unmixing matrix.</li>
	<li>Apply measured unmixing matrix for the whole stack.</li>
	<li>Save the new processed image stacks in *.tif format.</li>
</ol>

<ol>
	<li>Lumpkin, E. A., Caterina, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+sensory+transduction+in+the+skin.">Mechanisms of sensory transduction in the skin.</a> Nature. 445, 858-865 (2007).</li><li>Kennedy, W. R., Wendelschafer-Crabb, G., Johnson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+epidermal+nerves+in+diabetic+neuropathy.">Quantitation of epidermal nerves in diabetic neuropathy.</a> Neurology. 47, 1042-1048 (1996).</li><li>Wu, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morton's+interdigital+neuroma:+a+clinical+review+of+its+etiology,+treatment,+and+results.">Morton's interdigital neuroma: a clinical review of its etiology, treatment, and results.</a> J. Foot Ankle Surg. 35, 112-119 (1996).</li><li>Lauria, G., Lombardi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+biopsy:+a+new+tool+for+diagnosing+peripheral+neuropathy.">Skin biopsy: a new tool for diagnosing peripheral neuropathy.</a> BMJ. 334, 1159-1162 (2007).</li><li>Cheng, C., Guo, G. F., Martinez, J. A., Singh, V., Zochodne, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+plasticity+of+axons+within+a+cutaneous+milieu.">Dynamic plasticity of axons within a cutaneous milieu.</a> J. Neurosci. 30, 14735-14744 (2010).</li><li>Wang, B., Zinselmeyer, B. H., McDole, J. R., Gieselman, P. A., Miller, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+Imaging+of+Leukocyte+Homing+and+Migration+in+vivo.">Non-invasive Imaging of Leukocyte Homing and Migration in vivo.</a> J Vis Exp. (46), e2062 (2010).</li><li>Sacconi, L., O'Connor, R. P., Jasaitis, A., Masi, A., Buffelli, M., Pavone, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+multiphoton+nanosurgery+of+cortical+neurons.">In vivo multiphoton nanosurgery of cortical neurons.</a> J Biomed Opt. 12, 050502 (2007).</li><li>Robinson, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traumatic+injury+to+peripheral+nerves.">Traumatic injury to peripheral nerves.</a> Muscle Nerve. 23, 863-873 (2000).</li><li>Feng, G., Mellor, R. H., Bernstein, M., Keller-Peck, C., Nguyen, Q. T., Wallace, M., Nerbonne, J. M., Lichtman, J. W., Sanes, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neuronal+subsets+in+transgenic+mice+expressing+multiple+spectral+variants+of+GFP.">Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.</a> Neuron. 28, 41-51 (2000).</li><li>Marinkovic, P., Reuter, M. S., Brill, M. S., Godinho, L., Kerschensteiner, M., Misgeld, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+transport+deficits+and+degeneration+can+evolve+independently+in+mouse+models+of+amyotrophic+lateral+sclerosis.">Axonal transport deficits and degeneration can evolve independently in mouse models of amyotrophic lateral sclerosis.</a> Proc Natl Acad Sci U.S.A. 109, 4296-4301 (2012).</li><li>Amit, S., Yaron, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+systems+for+in+vivo+monitoring+and+microenvironmental+investigations+of+diabetic+neuropathy+in+a+murine+model.">Novel systems for in vivo monitoring and microenvironmental investigations of diabetic neuropathy in a murine model.</a> J Neural Transm. 119, 1317-1325 (2012).</li><li>Yu, H., Fischer, G., Jia, G., Reiser, J., Park, F., Hogan, Q. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+gene+transfer+into+the+dorsal+root+ganglion+of+adult+rats.">Lentiviral gene transfer into the dorsal root ganglion of adult rats.</a> Mol Pain. 7, 63 (2011).</li><li>Yuryev, M., Khiroug, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+longitudinal+investigation+of+individual+nerve+endings+in+the+skin+of+anesthetized+mice+using+in+vivo+two-photon+microscopy.">Dynamic longitudinal investigation of individual nerve endings in the skin of anesthetized mice using in vivo two-photon microscopy.</a> J Biomed Opt. 17, 046007 (2012).</li></ol>

The authors declare that they have no competing financial interests.

In this video protocol we demonstrate the method for non-invasive longitudinal two-photon imaging of single nerve endings.
The dynamics of skin innervations is affected in such diseases as psoriasis and peripheral neuropathy2, and in traumatic injuries9. Two-photon imaging allows detailed analysis of the nerve fibers structures in collagen matrix. The use of transgenic reporter mice helps to avoid the problems concerning the staining of the nerve fibers. Thy1-YFP-H strain seems to be sufficiently robust for morphological data analysis10 whereas the Thy1-mitoCFP mice may provide an opportunity of functional studies of mitochondrial dynamics in the nerve fibers11. The YFP-16/ICR line can be used for the observation of Meissner bodies12.&#160; The alternative approach may be the viral injection into dorsal root ganglion for the selective tracking of specific neuronal populations13.
It is worth noticing that the production of selective lesion within the collagen matrix is strongly hindered in comparison to the upper layers of skin. That is why sometimes exposure times for the dissection of nerve fibers can differ as much as tenfold while the intensity of the fluorescence for the imaging differs on the range of 10-20%. To maintain good quality of the image the constant immersion coupling has to be kept.
The resolution of the image usually depends on the desirable speed of acquisition. Typical time for production of a 30 frames- stack with resolution 800 x 800 pixels is 1 min, so one can decrease the resolution or thickness of imaged area to detect fast changes or increase resolution for fine morphological features studies.
Fixation of the footpad should be tight enough not to allow the drift of the image, but at the same time it should not affect the blood circulation in the skin. For optimization of the procedure, injections of blood vessels tracers (e.g., TexasRed labeled 70 kDa dextran) are possible, allowing one to adjust the plastic packaging material pressure via monitoring the blood flow. Motion artifacts in the preparation described in this protocol are unlikely because the animal is deeply anaesthetized, the paw itself is firmly attached to the fixation assembly, and because heartbeat and breathing movement are not directly transmitted to the paw. However, slight movements are possible especially if high magnification imaging is performed. In this case we can recommend using ImageJ plugins designed to compensate motion artifacts.
The repetitive imaging of the same area may be achieved by using such reference points as carpal pad14, or in case of injection of some substances in the footpad the spot of injection can serve as a landmark6. The other possibility is tattooing of the skin.

A correction was made to <a href="/video/51045">In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin</a>.
Leonard Khiroug was removed as an author.

Erratum: In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Cutaneous nerve endings undergo dynamical changes under different pathophysiological states. Nerve fibers can go through the process of degeneration and regeneration or restructuring in the course of such diseases as peripheral neuropathy2 or Morton&#8217;s neuroma3. After traumatic injury, an important part of nerve endings dynamics in skin is reinnervation of the damaged area. However, the common approach for investigation of nerve endings is ex vivo histological sectioning that lacks real-time information on the ongoing processes4. Using genetically encoded fluorescent markers, it is possible to track the nerve endings in skin of live animals, thus obtaining rich and significantly more relevant information on the structural changes. The investigation of cutaneous nerve endings is possible using conventional fluorescent microscopy, however, the strong light scattering of the skin tissue strongly undermines the quality of the data acquired5. Multiphoton microscopy allows acquisition of high-resolution images in the strongly scattering tissues due to the non-linear summation of energy of excitation light photons resulting in emission of fluorescence only from the focal point of the objective. This effect leads to a robust increase in the penetration depth and improvement of signal-to-noise ratio for the measurement in skin tissues6.&#160; Using the same laser as for imaging, it is possible to produce selective dissection of the nerve fibers7. In the following protocol we show the method of longitudinal imaging of cutaneous nerve endings in vivo in reporter transgenic mice combined with selective laser lesion using commercially available multiphoton microscope system.

<table border="0" cellpadding="0" cellspacing="0" width="1231">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Round cover glass&#160;</td>
			<td style="width:209px;">Electron Microscopy Science</td>
			<td style="width:119px;">72296-05</td>
			<td style="width:687px;">5 mm diameter #1.5 thickness</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Eye drops Viscotears</td>
			<td style="width:209px;">Novartis</td>
			<td style="width:119px;"></td>
			<td>2mg/g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Superglue Loctite 401</td>
			<td style="width:209px;">Henkel</td>
			<td>135429</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ketaminol</td>
			<td style="width:209px;">Intervet</td>
			<td style="width:119px;"></td>
			<td>Ketamine 50 mg/ml, working solution 10 mg/ml (use it 80 &#181;g per gramm of animal weight)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rompun</td>
			<td style="width:209px;">Bayer Healthcare</td>
			<td style="width:119px;"></td>
			<td>Xylazine 20 mg/ml, working solution 1.25 mg/ml (use it 10 &#181;g per gramm of animal weight)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:209px;"></td>
			<td style="width:119px;"></td>
			<td style="width:687px;">Working solution is a mixture of Ketamine 10 mg/ml and Xylazine 1.25 mg/ml in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol 70%</td>
			<td style="width:209px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Distilled water or Milli-Q water</td>
			<td style="width:209px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringes 1ml&#160;</td>
			<td style="width:209px;">BD</td>
			<td style="width:119px;">300013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">30G 1/2&#34; needles</td>
			<td style="width:209px;">BD</td>
			<td style="width:119px;">304000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Plastic packaging material</td>
			<td style="width:209px;"></td>
			<td style="width:119px;"></td>
			<td>Could be purchaized from general hardware store</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FV-1000MPE microscope</td>
			<td style="width:209px;">Olympus</td>
			<td style="width:119px;">FV-1000MPE</td>
			<td>Microscope with motorized stage and rigid metal bar for fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">25X XLPlan objective</td>
			<td style="width:209px;">Olympus</td>
			<td style="width:119px;">XLPLN 25XWMP</td>
			<td>Water imersion objective optimized for multiphoton imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mai-Tai DeepSee laser (2W)</td>
			<td style="width:209px;">SpectraPhysics</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Heating pad</td>
			<td style="width:209px;">Supertech</td>
			<td style="width:119px;">TMP-5b</td>
			<td>Heating pad with a temperature controller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Metal ring fixator</td>
			<td style="width:209px;">Neurotar Ltd.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageJ</td>
			<td style="width:209px;">NIH</td>
			<td style="width:119px;"></td>
			<td>Open source software for image processing and analysis, http://rsbweb.nih.gov/ij/&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Thy1-YFPH mice strain</td>
			<td style="width:209px;">JaxLab</td>
			<td style="width:119px;">003782</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo two-photon microscopy of single nerve endings in skin

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their close-to-surface positioning facilitates microscopic imaging of skin nerve endings in living intact animal. Using multiphoton microscopy, it is possible to obtain fine images overcoming the problem of strong light scattering of the skin tissue. Reporter transgenic mice that express EYFP under the control of Thy-1 promoter in neurons (including periphery sensory neurons) are well suited for the longitudinal studies of individual nerve endings over extended periods of time up to several months or even life-long. Furthermore, using the same femtosecond laser as for the imaging, it is possible to produce highly selective lesions of nerve fibers for the studies of the nerve fiber restructuring. Here, we present a simple and reliable protocol for longitudinal multiphoton in vivo imaging and laser-based microsurgery on mouse skin nerve endings.

Using the described technique it is possible to track the same fiber after the lesion and to study the degradation of the damaged nerve endings (Figure&#160;1). Acquisition of the stack with the thickness of 120-150 &#181;m is usually proper for the repetitive imaging during several days in order to keep the whole fiber in the field of view.
The lesion typically can be produced concisely when the plastic packaging material is adjusted to flatten the skin, so the collagen layers appear with uniform intensity on the image (Figure&#160;2). The nerve endings above the collagen layer are the most suitable to produce the precise lesion.
In case the skin is not flattened, the image may occur to be blurred (Figure&#160;3). The intensity in this situation can still be suitable for morphological investigations of the nerve fibers but the laser lesion procedure would be hindered.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51045/51045fig1highres.jpg" src="/files/ftp_upload/51045/51045fig1.jpg" /> 
Figure 1.&#160;Time track of the effect of laser lesion on the nerve fiber presented as maximum projection images. The upper images show the fiber before and directly after the lesion, the lower panel shows the same fiber after 30 min and 10 days (left and right panels, correspondingly). There are some YFP expressing structures of non-neuronal nature that appear 10 days after lesion. These structures are likely to be keratinocytes that are known to express Thy1 gene transiently under traumatic conditions. The YFP fluorescence of nerve fibers is shown in yellow. An arrow marks the region of the lesion. Scale bar 30 &#181;m.
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51045/51045fig2highres.jpg" src="/files/ftp_upload/51045/51045fig2.jpg" /> 
Figure 2.&#160;Maximum projection image for nerve endings in flattened skin area. The YFP fluorescence of nerve fibers is shown in yellow, the second harmonic generation of the collagen is shown in blue. Scale bar 50 &#181;m.
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/51045/51045fig3highres.jpg" src="/files/ftp_upload/51045/51045fig3.jpg" /> 
Figure 3.&#160;The nerve endings in the curved area of the skin obtained as a maximum z-projection for multiple optical sections. The YFP fluorescence of nerve fibers and autofluorescence of the hair is shown in yellow, the second harmonic generation of the collagen is shown in blue. Scale bar 50 &#181;m.
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/51045/51045fig4highres.jpg" src="/files/ftp_upload/51045/51045fig4.jpg" /> 
Figure 4.&#160;The paw fixation procedure using the microscope glass slide as a cover. The slide is attached to the metal bar using standard paper clips.

Watch this Scientific Journal Video about In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin at JoVE.com

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

The protocol for dynamic longitudinal imaging and selective laser lesion of nerve endings in reporter transgenic mice is presented.&#160;

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their ...

in-vivo-two-photon-microscopy-of-single-nerve-endings-in-skin

Nanyang Technological University

Research

JoVE Journal

Neuroscience

10.7K Views.  University of Helsinki. The protocol for dynamic longitudinal imaging and selective laser lesion of nerve endings in reporter transgenic mice is presented. 

Video: In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

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

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

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

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

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

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

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

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic neuroinflammatory state within the central nervous system (CNS), thought to be driven principally by virally-mediated activation of microglia and brain resident macrophages. HIV-1 infection is also accompanied by changes in cerebrovascular blood flow (CBF), raising the possibility that HIV-associated chronic neuroinflammation may lead to changes in CBF and/or in cerebral vascular architecture. To address this question, we have used a mouse model for HIV-induced neuroinflammation, and we have tested whether long-term exposure to this inflammatory environment may damage brain vasculature and result in rarefaction of capillary networks. In this paper we describe a method to quantify changes in cortical capillary density in a mouse model of neuroinflammatory disease (HIV-1 Tat transgenic mice). This generalizable approach employs in vivo two-photon imaging of cortical capillaries through a thin-skull cortical window, as well as ex vivo two-photon imaging of cortical capillaries in mouse brain sections. These procedures produce images and z-stack files of capillary networks, respectively, which can be then subjected to quantitative analysis in order to assess changes in cerebral vascular architecture.

This paper describes a method by which the vascular architecture in the brain can be quantified using in vivo and ex vivo two-photon microscopy.

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic ...

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles in the skin of the mouse pinna. The preparation is simple and relatively fast, reliably yielding extensive regions of multiple labeled units of living nerve terminals to study uptake and release of styryl pyridinium dyes extensively used in studies of vesicle recycling. Subdividing the preparations before labeling allows test vs. control comparisons in the same ear from a single individual. Helpful tips are given for improving the quality of the preparation, the labeling and the imaging parameters. This new system is suitable for assaying pharmacologically and mechanically-induced uptake and release of these vital dyes in lanceolate terminals in both wild-type and genetically modified animals. Examples of modulatory influences on labeling intensity are given.

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

Here we describe a novel preparation for imaging live lanceolate sensory terminals of palisade endings that innervate mouse ear skin hair follicles during staining and destaining with styryl pyridinium dyes.

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...