The authors thank T. Sato, M. Kanbayashi, and S. Kouyama for their technical assistance. This work was supported by JSPS KAKENHI Grant Numbers JP15K14322 and JP16H06143, the Takeda Science Foundation, the Uehara Memorial Foundation, and the Collaborative Research Project of Niigata University Brain Research Institute 2017-2923 (H.M.) and by KAKENHI JP16K14559, JP15H01454, and JP15H04263 and Grant-in Scientific Research on Innovation Areas &#34;Dynamic regulation of Brain Function by Scrap &#38; Build System&#34; (JP16H06459) from MEXT (T.I.).

Experiments should be performed in accordance with the animal welfare guidelines prescribed by the experimenter&#39;s institution.
1. Preparation of Pups for Imaging
NOTE: Pups with sparsely labeled cortical neurons can be obtained by in utero electroporation (IUE) of Supernova vectors5,6. The Supernova system consists of the following two vectors: TRE-Cre and CAG-loxP-STOP-loxP-Gene X-ires-tTA-WPRE. In this system, sparse labeling relies on TRE leakage. In a sparse population of transfected neurons, TRE drives the weak expression of Cre and tTA. Subsequently, only in these cells, the expression of gene X is facilitated by a positive feedback of the tTA-TRE cycles. The achieved sparse and bright labeling allows the visualization of morphological details of individual neurons in vivo. Details of the IUE procedure are not described in this protocol since they have been described elsewhere7,8,9,10,11.
<ol>
	<li>Prepare timed-pregnant mice for IUE.</li>
	<li>Prepare a DNA solution for IUE. For sparse labeling with RFP, use a solution containing pK031:TRE-Cre (5 ng/&#181;L) and pK029:CAG-loxP-STOP-loxP-RFP-ires-tTA-WPRE (1 &#181;g/&#181;L) or a solution containing pK031:TRE-Cre (5 ng/&#181;L) and pK273:CAG-loxP-STOP-loxP-CyRFP-ires-tTA-WPRE (1 &#181;g/&#181;L). 
	NOTE: Various proteins can be expressed in the labeled neurons using different combinations of vectors. Also, various genes can be knocked-down or knocked-out specifically in labeled cells5,6 (e.g., a series of vectors for the Supernova system are available from RIKEN BioResource Research Center and from Addgene).</li>
	<li>Perform regular IUE7,8,9,10,11 to label cortical neurons. For the labeling of layer 4 neurons, use embryonic day-14 embryos.</li>
	<li>Wait for pup delivery and growth.</li>
</ol>
2. Surgery
<ol>
	<li>Anesthetize the postnatal day-5 (P5) pup using isoflurane gas (1.0%). Perform a tail-pinch test to check the level of anesthesia. If the pup responds to the pinch, increase the isoflurane concentration (up to 2.0%) or wait until the response disappears. Maintain the pup&#39;s body temperature during surgery using a heating pad.</li>
	<li>Subcutaneously inject an analgesic (carprofen, 5 mg/kg).</li>
	<li>Sterilize the pup&#39;s skin covering the skull by wiping it with 70% ethanol three times.</li>
	<li>Remove approximately 20 mm2 of the skin covering the skull using scissors sterilized with 70% ethanol (Figure 1A).</li>
	<li>Remove the fascia of the skull using sterilized forceps and a clean, sterile cotton swab (Figure 1A).</li>
	<li>Apply tissue adhesive using loading tips to the incised skin surface to stop the bleeding. Do not apply tissue adhesive to the imaging area (Figure 1B) because this makes the opening of the skull more difficult.</li>
	<li>Place the pup on a heating pad (37 &#176;C) and allow it to recover from anesthesia. Wait until the tissue adhesive has dried and solidified (approximately 30 min).</li>
	<li>If necessary, apply more tissue adhesive and wait for it to dry and solidify.</li>
</ol>
3. Cranial Window Preparation
<ol>
	<li>Anesthetize the pup using isoflurane (1.0% - 2.0%) and check the anesthesia level by a tail-pinch test.</li>
	<li>Carefully open the skull with a sterilized razor blade leaving the dura intact (1 mm in diameter) (Figure 1C). Use a gelatin sponge (cut into small pieces, approximately &#60; 2 mm3, using sterilized scissors, and apply them using tweezers) soaked in cortex buffer12 (125 mmol/L NaCl, 5 mmol/L KCl, 10 mmol/L glucose, 10 mmol/L HEPES, 2 mmol/L CaCl2, and 2 mmol/L MgSO4; pH 7.4; 300 mOsm/L; room temperature) to stop the bleeding. When opening the skull, apply a cortex buffer to keep the brain surface moist.</li>
	<li>Remove any buffer and blood from the dural surface using a gelatin sponge. Apply a thin layer of 1.0% low-melting-point agarose (dissolved in cortex buffer) using yellow tips. Using a heat block machine, maintain the temperature of the agarose solution at 42&#176;C until application. 
	NOTE: The draining of buffer and blood must be performed from the side of the craniotomy while taking care that the dry gelatin sponge does not come in contact with the dura. Failure to do so may damage the dura.</li>
	<li>Apply a round glass coverslip (No. 1, 3 mm in diameter) onto the agarose gel layer. Remove all bubbles between the coverslip and the agarose gel layer by pouring an excess of agarose gel between them. Remove the excess gel protruding from under the coverslip using tweezers (Figure 1D and 1E).</li>
	<li>Secure the coverslip using dental cement (Figure 1F).</li>
	<li>Mix the cement powder and the cement liquid. Apply the mixture using yellow tips before it becomes solidified. Do not apply dental cement onto the dura, because this may damage the brain.</li>
	<li>Attach a sterile titanium bar (custom made, approximately 30 mg, see Figure 1G) on the cranial bone using dental cement. Align the titanium bar and the coverslip (on the surface of the dura) in parallel to easily capture images.</li>
	<li>Cover the exposed skull with dental cement (Figure 1H).</li>
	<li>Recover the pup from anesthesia. Keep it on a heater (37 &#176;C) until the dental cement has solidified (1 h).</li>
</ol>
4. Two-photon Imaging
NOTE: The in vivo images in Figure 2 were acquired using a two-photon microscope with a titanium-sapphire laser (beam diameter [1/e2]2: 1.2 mm).
<ol>
	<li>Set the two-photon laser wavelength. For RFP excitation, use 1,000 nm (450 mW/mm2 at 400 &#181;m of depth). 
	NOTE: The laser power should be reduced as the z-position moves up.</li>
	<li>Wipe the surface of the coverslip with 70% ethanol.</li>
	<li>Anesthetize the pup using isoflurane (1.5% - 2.0%) and check the anesthesia level using a tail-pinch test.</li>
	<li>Attach the pup to the titanium plate on the imaging stage using a titanium bar on the pup&#39;s head (Figure 2A and 2B). Adjust the head such that the coverslip is parallel to the objective lens using the goniometer stage (Figure 2B). Maintain body temperature of the pup using a heating pad (37 &#176;C).</li>
	<li>Set the isoflurane concentration to 0.7% - 1.0%. 
	NOTE: A very high isoflurane concentration may cause accidental death of the pup during imaging.</li>
	<li>Place the imaging stage under the objective lens (20X, NA 1.0) of the two-photon microscope (Figure 2B and 2C).</li>
	<li>Apply one drop of water onto the coverslip. Use epi-fluorescence to locate the fluorescent protein-labeled neurons in the area where the dura has been exposed.</li>
	<li>Acquire z-stack images at 1.4-&#181;m intervals. For layer 4 neuron imaging, set the z-width to 150 - 300 &#181;m to image the entire dendritic morphology (Figure 2D and 2E). Use slow scanning and averaging to get clear images showing the neuronal morphology (it usually takes &#62; 20 min to acquire the entire dendritic morphology). 
	NOTE: The following parameters are recommended for imaging. Excitation wavelength: 1,000 nm, scanner: galvanometer type, dichroic mirror: 690 nm, emission filter: 575 - 620 nm bandpass, detector: GaAsP type, gain setting &#62; 100, image size &#62; 512 x 512 &#181;m, field of view &#62; 600 x 600 &#181;m, pixel resolution &#60; 1.2 &#181;m.</li>
</ol>
5. Recovery and Nursing
<ol>
	<li>Detach the pup from the imaging stage.</li>
	<li>Place the pup on a heater (37 &#176;C) and allow it to recover (15 min).</li>
	<li>Feed the pup warm milk using a micropipette at 2-h intervals and gently stimulate the stomach to allow excretion. Confirm that the pup is drinking milk by measuring its body weight.</li>
</ol>
6. Re-imaging
<ol>
	<li>Anesthetize the pup with isoflurane (1.5% - 2.0%), and check the anesthesia level using a tail-pinch test. Attach it to the imaging stage.</li>
	<li>Locate the previously imaged neurons and acquire a z-stack image. The identification of neurons is easy owing to their sparse labeling with Supernova.</li>
	<li>Repeat steps 5.1 - 6.2 until imaging is completed.&#160;NOTE: The pup can be imaged up to 18 h without losing weight. 
	</li>
</ol>

<ol><li>Lendvai, B., Stern, E. A., Chen, B., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Experience-dependent+plasticity+of+dendritic+spines+in+the+developing+rat+barrel+cortex+in+vivo%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Experience-dependent plasticity of dendritic spines in the developing rat barrel cortex in vivo.</a> Nature. 404 (6780), 876-881 (2000).</li>
<li>Grutzendler, J., Kasthuri, N., Gan, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+dendritic+spine+stability+in+the+adult+cortex%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Long-term dendritic spine stability in the adult cortex.</a> Nature. 420 (6917), 812-816 (2002).</li>
<li>Carrillo, J., Nishiyama, N., Nishiyama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dendritic+translocation+establishes+the+winner+in+cerebellar+climbing+fiber+synapse+elimination%5BTitle%5D)+AND+%22The+Journal+of+Neuroscience%22%5BJournal%5D)">Dendritic translocation establishes the winner in cerebellar climbing fiber synapse elimination.</a> The Journal of Neuroscience. 33 (18), 7641-7653 (2013).</li>
<li>Portera-Cailliau, C., Weimer, R. M., De Paola, V., Caroni, P., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diverse+modes+of+axon+elaboration+in+the+developing+neocortex%5BTitle%5D)+AND+%22PLoS+Biology%22%5BJournal%5D)">Diverse modes of axon elaboration in the developing neocortex.</a> PLoS Biology. , e272(2005).</li>
<li>Mizuno, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NMDAR-regulated+dynamics+of+layer+4+neuronal+dendrites+during+thalamocortical+reorganization+in+neonates%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">NMDAR-regulated dynamics of layer 4 neuronal dendrites during thalamocortical reorganization in neonates.</a> Neuron. 82 (2), 365-379 (2014).</li>
<li>Luo, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Supernova%3A+A+Versatile+Vector+System+for+Single-Cell+Labeling+and+Gene+Function+Studies+in+vivo%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Supernova: A Versatile Vector System for Single-Cell Labeling and Gene Function Studies in vivo.</a> Scientific Reports. 6, 35747(2016).</li>
<li>Mizuno, H., Hirano, T., Tagawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evidence+for+activity-dependent+cortical+wiring%3A+formation+of+interhemispheric+connections+in+neonatal+mouse+visual+cortex+requires+projection+neuron+activity%5BTitle%5D)+AND+%22The+Journal+of+Neuroscience%22%5BJournal%5D)">Evidence for activity-dependent cortical wiring: formation of interhemispheric connections in neonatal mouse visual cortex requires projection neuron activity.</a> The Journal of Neuroscience. 27 (25), 6760-6770 (2007).</li>
<li>Saito, T., Nakatsuji, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+gene+transfer+into+the+embryonic+mouse+brain+using+in+vivo+electroporation%5BTitle%5D)+AND+%22Developmental+Biology%22%5BJournal%5D)">Efficient gene transfer into the embryonic mouse brain using in vivo electroporation.</a> Developmental Biology. 240 (1), 237-246 (2001).</li>
<li>Tabata, H., Nakajima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+in+utero+gene+transfer+system+to+the+developing+mouse+brain+using+electroporation%3A+visualization+of+neuronal+migration+in+the+developing+cortex%5BTitle%5D)+AND+%22Neuroscience%22%5BJournal%5D)">Efficient in utero gene transfer system to the developing mouse brain using electroporation: visualization of neuronal migration in the developing cortex.</a> Neuroscience. 103 (4), 865-872 (2001).</li>
<li>Mizuno, H., Hirano, T., Tagawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pre-synaptic+and+post-synaptic+neuronal+activity+supports+the+axon+development+of+callosal+projection+neurons+during+different+post-natal+periods+in+the+mouse+cerebral+cortex%5BTitle%5D)+AND+%22European+Journal+of+Neuroscience%22%5BJournal%5D)">Pre-synaptic and post-synaptic neuronal activity supports the axon development of callosal projection neurons during different post-natal periods in the mouse cerebral cortex.</a> European Journal of Neuroscience. 31 (3), 410-424 (2010).</li>
<li>Matsui, A., Yoshida, A. C., Kubota, M., Ogawa, M., Shimogori, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+in+utero+electroporation%3A+controlled+spatiotemporal+gene+transfection%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Mouse in utero electroporation: controlled spatiotemporal gene transfection.</a> Journal of Visualized Experiments. (54), e3024(2011).</li>
<li>Holtmaat, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term%2C+high-resolution+imaging+in+the+mouse+neocortex+through+a+chronic+cranial+window%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window.</a> Nature Protocols. 4 (8), 1128-1144 (2009).</li>
<li>Nakai, J., Ohkura, M., Imoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+high+signal-to-noise+Ca%282%2B%29+probe+composed+of+a+single+green+fluorescent+protein%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein.</a> Nature Biotechnology. 19 (2), 137-141 (2001).</li>
<li>Chen, T. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ultrasensitive+fluorescent+proteins+for+imaging+neuronal+activity%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Ultrasensitive fluorescent proteins for imaging neuronal activity.</a> Nature. 499 (7458), 295-300 (2013).</li>
<li>Mizuno, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Patchwork-Type+Spontaneous+Activity+in+Neonatal+Barrel+Cortex+Layer+4+Transmitted+via+Thalamocortical+Projections%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">Patchwork-Type Spontaneous Activity in Neonatal Barrel Cortex Layer 4 Transmitted via Thalamocortical Projections.</a> Cell Reports. 22 (1), 123-135 (2018).</li>
<li>Zong, H., Espinosa, J. S., Su, H. H., Muzumdar, M. D., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mosaic+analysis+with+double+markers+in+mice%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Mosaic analysis with double markers in mice.</a> Cell. 121 (3), 479-492 (2005).</li>
<li>Young, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-neuron+labeling+with+inducible+Cre-mediated+knockout+in+transgenic+mice%5BTitle%5D)+AND+%22Nature+Neuroscience%22%5BJournal%5D)">Single-neuron labeling with inducible Cre-mediated knockout in transgenic mice.</a> Nature Neuroscience. 11 (6), 721-728 (2008).</li>
<li>Liu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neonatal+Repeated+Exposure+to+Isoflurane+not+Sevoflurane+in+Mice+Reversibly+Impaired+Spatial+Cognition+at+Juvenile-Age%5BTitle%5D)+AND+%22Neurochemical+Research%22%5BJournal%5D)">Neonatal Repeated Exposure to Isoflurane not Sevoflurane in Mice Reversibly Impaired Spatial Cognition at Juvenile-Age.</a> Neurochemical Research. 42 (2), 595-605 (2017).</li>
<li>Kondo, M., Kobayashi, K., Ohkura, M., Nakai, J., Matsuzaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Two-photon+calcium+imaging+of+the+medial+prefrontal+cortex+and+hippocampus+without+cortical+invasion%5BTitle%5D)+AND+%22eLIFE%22%5BJournal%5D)">Two-photon calcium imaging of the medial prefrontal cortex and hippocampus without cortical invasion.</a> eLIFE. 6, e26839(2017).</li>
<li>Nakazawa, S., Mizuno, H., Iwasato, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+dynamics+of+cortical+neuron+dendritic+trees+revealed+by+long-term+in+vivo+imaging+in+neonates%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Differential dynamics of cortical neuron dendritic trees revealed by long-term in vivo imaging in neonates.</a> Nature Communications. 9 (1), 3106(2018).</li>
</ol>

The authors have nothing to disclose.

Critical Steps in the Protocol and Troubleshooting:
The most critical step of the protocol is the removal of the skull (Protocol step 3.2). Upon insertion, the razor blade often adheres to the dura, causing dural bleeding and damage to the brain. This can be avoided by adding a drop of cortex buffer on the skull and removing the skull in cortex buffer.
Bleeding from the dura and the skin after cranial window preparation leads to occlusion of the window. To avoid this, the tissue adhesive and dental cement used should be allowed to completely dry before proceeding to the next step. Multiple pups with a cranial window should be prepared since it is difficult to completely avoid bleeding. In general, if the cranial window remains clear and stable for 2 - 3 h post-surgery, the pup can be used for time-lapse imaging.
Significance of the Method with Respect to Existing/Alternative Methods:
Here, we have described a method for in vivo two-photon imaging of the neonatal cortex. This protocol has several merits compared with previously reported methods. These are listed as follows.
1) Cortical neurons can be sparsely and brightly labeled by transfecting Supernova vectors using IUE. IUE has been widely used for the labeling of cortical neurons during developing stages. However, a simple IUE is unsuitable for the imaging of individual neurons since neurons may be labeled too densely7,8,9,10. Moreover, using the Supernova system, various types of fluorescent proteins can be used for labeling sparse populations of neurons. For example, using Supernova-mediated sparse labeling of a genetically encoded calcium indicator GCaMP13,14, we have recently performed a functional analysis of individual neurons in the developing cortex layer 4 (P3 to P13)15.
2) The method described in this study is suitable for elucidating the molecular mechanisms underlying neuronal circuit development. The Supernova system allows sparsely labeled cell-specific knockout of any gene. Thus, the dynamics of a neuron containing a specific gene disruption can be observed. For this purpose, genetics-based systems such as MADM16 and SLICK17 have been previously reported. However, because the breeding of mouse lines is essential for these systems, they require much time and cost than the protocol described here.
3) This study utilizes a razor blade for skull removal. This minimizes bleeding from the dura and allows the opening of a wide area of the skull (&#60; 2 mm in diameter). Using this procedure, it was possible to observe the spontaneous activity of layer 4 neurons in the entire large barrel area within the somatosensory cortex15.
Limitations of the method:
A disadvantage of in vivo imaging of the mouse neonatal brain, compared with the imaging of transparent animals such as zebrafish larvae and Xenopus tadpoles, is the lower spatial and temporal resolution. Slow scanning and averaging should be performed for yielding clear images of the neuronal morphology because more light scattering occurs in the mouse brain. Light scattering may possibly be reduced using a longer wavelength laser for fluorescent protein excitation and proteins with longer fluorescence emission wavelengths.
Another limitation of the present protocol could be that the surgery for cranial window implantation may affect the formation of a normal cortical circuit due to brain inflammation12. However, there is a high likelihood that in vivo imaging is more physiological compared with in vitro imaging such as time-lapse imaging of the brain slice preparation, which should also give rise to severe inflammation by ischemia and slicing. It has also been reported that repetitive exposure to isoflurane may affect several neuronal processes18. Control experiments should be performed for verifying the appropriateness of results obtained by in vivo imaging. In the case of our imaging of layer 4 neurons in the somatosensory cortex, we confirmed a normal increase in total dendritic length and acquisition of orientation bias of dendritic projections of spiny stellate neurons5,20.
Recent studies reported &#62; 1-mm-depth imaging in an adult mouse brain wherein the dura was removed during surgery19. On the other hand, we have been able to report up to 400-&#181;m-depth imaging in neonates5. Since the dura cannot be removed in neonates and this, in turn, leads to a high light scattering, we consider that deep imaging in neonates is more challenging than in adults. Future improvement in fluorescent probes, lasers, and detectors should allow deep imaging in neonatal brains.
Thus far, we have reported 18-hour time-lapse imaging using the present protocol5. Recently, we have succeeded in 72-hour time-lapse imaging by improving the protocol20. We will continue to refine the protocol presented here (e.g., longer-term, higher time, and/or spatial resolution imaging) for revealing dynamic mechanisms of neuronal circuit development.

The precise wiring of neuronal circuits in the cerebral cortex is essential for higher brain functions including perception, cognition, and learning and memory. Cortical circuits are dynamically refined during postnatal development. Studies have investigated the process of cortical circuit formation using histological and in vitro culture analyses. However, the dynamics of circuit formation in living mammals has remained mostly unexplored.
Two-photon microscopy has been widely used for the in vivo analyses of neuronal circuits in the adult mouse brain1,2. However, owing to technical challenges, only a limited number of studies have addressed neuronal circuit formation in newborn mice. For example, Carrillo et al. performed the time-lapse imaging of climbing fibers in the cerebellum in the second postnatal week3. Portera-Cailliau et al. reported the imaging of axons in cortical layer 1 in the first postnatal week4. In the present study, we summarize a protocol for the observation of layer 4 cortical neurons and their dendrites in newborn mice. Results obtained by applying this protocol, which includes two methodologies, are reported in our recent publication5. First, we use the Supernova vector system5,6 for labeling individual neurons in the neonatal brain. In the Supernova system, the fluorescent proteins used for neuronal labeling are exchangeable and labeled cell-specific gene knockdown and editing/knockout analyses are also possible. Second, we describe a surgical procedure for cranial window preparation in fragile neonatal mice. Together, these methodologies allow the in vivo observation of individual neurons in neonatal brains.

<table><tbody><tr><td>pK031. TRE-Cre</td><td>Authors</td><td>-</td><td>Available from RIKEN BRC and Addgene</td></tr><tr><td>pK029. CAG-loxP-STOP-loxP-RFP-ires-tTA-WPRE</td><td>Authors</td><td>-</td><td>Available from RIKEN BRC and Addgene</td></tr><tr><td>pK273. CAG-loxP-STOP-loxP-CyRFP-ires-tTA-WPRE</td><td>Authors</td><td>-</td><td>Available from authors</td></tr><tr><td>Isoflurane</td><td>Wako</td><td>099-06571</td><td></td></tr><tr><td>410 Anaesthesia Unit (isoflurane gas machine)</td><td>Univentor</td><td>8323101</td><td></td></tr><tr><td>Vetbond (tissue adhesive)</td><td>3M</td><td>084-1469SB</td><td></td></tr><tr><td>M&amp;micro;ltiFlex Round (loading tip)</td><td>Sorenson</td><td>13810</td><td></td></tr><tr><td>Gelfoam (gelatin sponge)</td><td>Pfizer</td><td>09-0353-01</td><td></td></tr><tr><td>Agarose</td><td>Sigma</td><td>A9793</td><td>Low melting point</td></tr><tr><td>Round-shaped coverslip</td><td>Matsunami</td><td>-</td><td>Custom made</td></tr><tr><td>Unifast 2 (dental cement)</td><td>GC</td><td>-</td><td></td></tr><tr><td>Titanium bar</td><td>Authors</td><td>-</td><td>Custom made (see Figure 1G)</td></tr><tr><td>Rimadyl (carprofen)</td><td>Zoetis</td><td>-</td><td>Injectable</td></tr><tr><td>2-photon microscope</td><td>Zeiss</td><td>LSM7MP</td><td></td></tr><tr><td>Titanium-sapphire laser</td><td>Spertra-Physics</td><td>Mai-Tai eHPDS</td><td></td></tr><tr><td>Titanium plate</td><td>Authors</td><td>-</td><td>Custom made (see Figure 2A)</td></tr><tr><td>IMARIS, FilamentTracer, MeasurementPro</td><td>BITPLANE</td><td></td><td></td></tr><tr><td>Goniometer stage</td><td>Thorlabs</td><td>GN2/M</td><td></td></tr></tbody></table>

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.

Figures 2D - 2F show representative results of two-photon time-lapse imaging of layer 4 cortical neurons using the present protocol. For the purpose of analysis, select neurons with clear dendritic morphology throughout the imaging periods. We analyzed the dendritic morphology of imaged neurons using morphological analysis software. Representative dendritic morphology reconstruction is shown in Figure 2F. Neurons showing disconnected dendrites (Figure 2G) should be excluded from analyses, because disconnected dendrites indicate cell death induced by damage during surgery or imaging. In addition, neurons with blurred dendritic tips should be excluded (e.g., the neuron with the arrowhead in Figure 2D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58340/58340fig1.jpg" /> 
Figure 1: Surgery, cranial window preparation, and attachment of the titanium bar. (A) This panel shows the removal of the skin covering the skull. (B) This panel shows the fixation of the gap between the skin and the skull. Be careful not to apply the bond to the imaging area. (C) This is an image of the exposed dura. A razor blade was inserted between the skull and the dura, and the skull was flapped open to the left of the exposed area (arrowhead). The bone can then be easily removed using forceps. (D) This is a schematic design showing a vertical view of the cranial window. The gap between the cover glass and the dura is filled with a thin layer of agarose gel. The coverslip is fixed to the skull using dental cement. (E) A round-shaped coverslip is placed on the agarose gel layer. (F) This is an image of the secured coverslip. (G) This panel shows the design of the titanium bar. The titanium bar contains two screw holes for attachment to the imaging stage (see Figure 2) and one flat rectangular part that is attached to the pup&#39;s head. (H) The rectangular part of the titanium bar is attached to the pup&#39;s skull using dental cement. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58340/58340fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58340/58340fig2.jpg" /> 
Figure 2: In vivo two-photon imaging of cortical neurons in neonatal mice. (A) This panel shows the design of the titanium plate. The titanium plate has two screw holes for attaching the titanium bar and four screw holes for attaching the goniometer stage, which is placed on the imaging stage. 2.5 mm (in diameter) x 2 mm (in length) screws are used for fixation. (B) This is a representative image of the pup attached to the imaging stage. The pup&#39;s body temperature is maintained using a heater. (C) The pup is anesthetized using isoflurane during the in vivo imaging procedure. (D) This panel shows a representative Z-stack time-lapse image of layer 4 cortical neurons of a P5 pup. The arrowhead indicates the neuron with blurred dendrites, which should be removed from the analysis. (E) This panel shows higher magnification time-lapse images of the neuron with arrows in panel D. Blue arrowheads: dendritic tips that are retracted in 4.5 h, yellow arrowheads: dendritic tips that are elongated in 4.5 h, small white arrowheads: axon of a neighboring cell. (F) This is a 3-D model of the dendritic trace of neuron in the left figure of panel E. Blue circles indicate the position of the cell body. (G) This panel shows representative neurons with disconnected dendrites, which are not included in the analysis. The sample data in panels D - G contain NR1 (an essential subunit of NMDA-type glutamate receptor)-knocked-out neurons (because of limited data available from the authors). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58340/58340fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice at JoVE.com

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.

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 ...

in-vivo-two-photon-imaging-of-cortical-neurons-in-neonatal-mice

Nanyang Technological University

Research

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Neuroscience

12.1K Views.  Kumamoto University. 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.

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

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

Biology

Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging

In the primary visual cortex of non-rodent mammals, neurons are clustered according to their preference for stimulus features such as orientation1-4, direction5-7, ocular dominance8,9 and binocular disparity9. Orientation selectivity is the most widely studied feature and a continuous map with a quasi-periodic layout for preferred orientation is present across the entire primary visual cortex10,11. Integrating the synaptic, cellular and network contributions that lead to stimulus selective responses in these functional maps requires the hybridization of imaging techniques that span sub-micron to millimeter spatial scales. With conventional intrinsic signal optical imaging, the overall layout of functional maps across the entire surface of the visual cortex can be determined12. The development of in vivo two-photon microscopy using calcium sensitive dyes enables one to determine the synaptic input arriving at individual dendritic spines13 or record activity simultaneously from hundreds of individual neuronal cell bodies6,14. Consequently, combining intrinsic signal imaging with the sub-micron spatial resolution of two-photon microscopy offers the possibility of determining exactly which dendritic segments and cells contribute to the micro-domain of any functional map in the neocortex. Here we demonstrate a high-yield method for rapidly obtaining a cortical orientation map and targeting a specific micro-domain in this functional map for labeling neurons with fluorescent dyes in a non-rodent mammal. With the same microscope used for two-photon imaging, we first generate an orientation map using intrinsic signal optical imaging. Then we show how to target a micro-domain of interest using a micropipette loaded with dye to either label a population of neuronal cell bodies or label a single neuron such that dendrites, spines and axons are visible in vivo. Our refinements over previous methods facilitate an examination of neuronal structure-function relationships with sub-cellular resolution in the framework of neocortical functional architectures.

A method is described for labeling neurons with fluorescent dyes in predetermined functional micro-domains of the neocortex. First, intrinsic signal optical imaging is used to obtain a functional map. Then two-photon microscopy is used to label and image neurons within a micro-domain of the map.

In the primary visual cortex of non-rodent mammals, neurons are clustered according to their preference for stimulus features such as orientation1-4, ...

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 ...

Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. Although studying these cells in isolation in vitro or together ex vivo provides useful physiological information; salient features of neural cell physiology will be missed in such contexts. Therefore, there is a need for studying neural cells in their native in vivo environment. The protocol detailed here describes repetitive in vivo two-photon imaging of neural cells in the rodent cortex as a tool to visualize and study specific cells over extended periods of time from hours to months. We describe in detail the use of the grossly stable brain vasculature as a coarse map or fluorescently labeled dendrites as a fine map of select brain regions of interest. Using these maps as a visual key, we show how neural cells can be precisely relocated for subsequent repetitive in vivo imaging. Using examples of in vivo imaging of fluorescently-labeled microglia, neurons, and NG2+ cells, this protocol demonstrates the ability of this technique to allow repetitive visualization of cellular dynamics in the same brain location over extended time periods, that can further aid in understanding the structural and functional responses of these cells in normal physiology or following pathological insults. Where necessary, this approach can be coupled to functional imaging of neural cells, e.g., with calcium imaging. This approach is especially a powerful technique to visualize the physical interaction between different cell types of the CNS in vivo when genetic mouse models or specific dyes with distinct fluorescent tags to label the cells of interest are available.

This protocol describes a chronic cranial window implantation technique that can be used for longitudinal imaging of neuro-glio-vascular structures, interactions, and function in both healthy and diseased conditions. It serves as a complementary alternative to the transcranial imaging approach that, while often preferred, possesses some critical limitations.

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. ...

Immunology and Infection

Neuronal Activity Mapping in Developing Mouse Sensory Cortex

In Vivo Visualization of Spontaneous Activity in Neonatal Mouse Sensory Cortex at a Single-Neuron Resolution

The mammalian brain undergoes dynamic developmental changes at both the cellular and circuit levels throughout prenatal and postnatal periods. Following the discovery of numerous genes contributing to these developmental changes, it is now known that neuronal activity also substantially modulates these processes. In the developing cerebral cortex, neurons exhibit synchronized activity patterns that are specialized to each primary sensory area. These patterns markedly differ from those observed in the mature cortex, emphasizing their role in regulating area-specific developmental processes. Deficiencies in neuronal activity during development can lead to various brain diseases. These findings highlight the need to examine the regulatory mechanisms underlying activity patterns in neuronal development. This paper summarizes a series of protocols to visualize primary sensory areas and neuronal activity in neonatal mice, to image the activity of individual neurons within the cortical subfields using two-photon microscopy in vivo, and to analyze subfield-related activity correlations. We show representative results of patchwork-like synchronous activity within individual barrels in the somatosensory cortex. We also discuss various potential applications and some limitations of this protocol.

Author Spotlight: Deciphering Neural Circuit Formation from Two-Photon Microscopy and Single Neuron Imaging

Primary sensory areas in the neocortex exhibit unique spontaneous activities during development. This article describes how to visualize individual neuron activities and primary sensory areas to analyze area-specific synchronous activities in neonatal mice in vivo.

The mammalian brain undergoes dynamic developmental changes at both the cellular and circuit levels throughout prenatal and postnatal periods. Following ...

 Two-Photon Microscopy for In Vivo Imaging of Mouse Brain Microvasculature 

Source:<a style='text-decoration:none;' href='https://app-jove-com.remotexs.ntu.edu.sg/v/53582/quantification-of-cerebral-vascular-architecture-using-two-photon-microscopy-in-a-mouse-model-of-hiv-induced-neuroinflammation'><span style='-webkit-text-decoration-skip:none;font-family:Roboto,sans-serif;font-size:10pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>&#160;<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>Nishimura, C.&#160;et al.<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'> Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation.&#160;J. Vis. Exp.<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'> (2016).</a> &#160;This video demonstrates the in vivo visualization of cortical capillaries in the mouse brain using two-photon microscopy. It outlines the steps for injecting a fluorescent dye into an anesthetized mouse with a thin-skull cortical window, preparing the setup for imaging, and acquiring detailed images of brain microvascular structures for analysis.

Source:&#160;Nishimura, C.&#160;et al. Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced ...

JoVE Encyclopedia of Experiments

Neuroimaging

Neurovascular & Blood Flow Imaging

Two-Photon In Vivo Imaging of the Mouse Retina

<a href='https://app-jove-com.remotexs.ntu.edu.sg/v/61970/transpupillary-two-photon-in-vivo-imaging-of-the-mouse-retina'>Source: Wang, Z., et al.&#160;Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina. J. Vis. Exp.&#160;(2021).</a>This video demonstrates a technique for in vivo retinal imaging using two-photon microscopy. Anesthetized mice are prepared for imaging by securing their heads, Applying eye lubricant, and placing a coverslip over the eye. Retinal ganglion cells (RGCs) expressing fluorescent proteins are first visualized using epifluorescence for alignment. Finally, two-photon imaging is employed, allowing for high-resolution visualization of RGCs.

Source: Wang, Z., et al.&#160;Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina. J. Vis. Exp.&#160;(2021).This video demonstrates a technique ...

Live Imaging & In Vivo Tracking

Two-Photon Microscopy for Imaging Neuronal Morphology  in a Mouse Pup

Source:&#160;<a href='https://app-jove-com.remotexs.ntu.edu.sg/v/58340/in-vivo-two-photon-imaging-of-cortical-neurons-in-neonatal-mice'>Mizuno, H.,&#160;et al.&#160;In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice.&#160;J. Vis. Exp. (2018)</a>This video showcases two-photon microscopy being used to image neurons in the brain of an anesthetized transgenic mouse pup. The two-photon laser allows for deeper imaging with high spatial resolution and minimal light scattering, enabling the detailed capture of neuronal morphology, including dendritic projections.

Source:&#160;Mizuno, H.,&#160;et al.&#160;In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice.&#160;J. Vis. Exp. (2018)This video showcases ...

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

Podsumowanie

Przeglądaj więcej filmów

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging

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

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

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

Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice

In Vivo Visualization of Spontaneous Activity in Neonatal Mouse Sensory Cortex at a Single-Neuron Resolution

Two-Photon Microscopy for In Vivo Imaging of Mouse Brain Microvasculature

Two-Photon In Vivo Imaging of the Mouse Retina

Two-Photon Microscopy for Imaging Neuronal Morphology in a Mouse Pup