This study was supported by the Lundbeck Foundation, the NOVO-Nordisk Foundation, the Danish Council for Independent Research | Medical Sciences, and the NORDEA Foundation Grant to the Center for Healthy Aging.

All procedures involving animals were approved by the Danish National Ethics Committee according to the guidelines set forth in the European Council&#8217;s Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes and were in compliance with the ARRIVE guidelines.&#160;This is a terminal procedure with the mice being euthanized prior to anesthetic recovery.
1. Pre-surgical preparation
<ol>
	<li>Clean the surgical table and all the surrounding area with 70% ethanol. Thoroughly clean and dry the surgical tools before the surgery.</li>
	<li>Anesthetize an NG2DsRed mouse (Tg(Cspg4-DsRed.T1)1Akik/J; both genders; 4-8 months old) by a peritoneal injection of ketamine + xylazine (60 mg/kg + 10 mg/kg) dissolved in sterilized water, pH 7.4. Administer supplemental doses of ketamine (30 mg/kg) every 25 min until completion of the surgical procedure. Check tail and toe pinch reflexes regularly to verify the depth of anesthesia.</li>
	<li>Inject saline subcutaneously at four different locations on the back of the mouse for a total volume of 1 mL to prevent dehydration during the experiment. To protect the eyes from drying out, apply eye lubricant. Maintain body temperature at 37 &#176;C using a rectal temperature probe and heating blanket.</li>
</ol>
2. Surgical procedure
<ol>
	<li>Tracheotomy 
	<ol>
		<li>Place the mouse on its back. Inject 0.04 mL of 0.5% lidocaine subcutaneously under the planned incision and wait for 2 min. Make a 10 mm long incision above the chest bone (manubrium). Separate the submandibular glands and sternothyroid muscles, and then expose the trachea.</li>
		<li>Make tracheostomy between two tracheal rings with scissors. Insert a small metal tube with a length of 16.2 mm and a diameter of 1 mm into the trachea. Connect the tube to a mechanical ventilator and capnograph to monitor the end-expiratory CO2 (Figure 1A).</li>
	</ol>
	</li>
	<li>Catheter insertion
	<ol>
		<li>Inject 0.5% lidocaine (0.04 mL) subcutaneously under the planned incision and wait for 2 min.</li>
		<li>Using fine tip forceps gently separate the artery from the vein. Stop the blood flow upstream with a microvascular clamp. Constrict the blood flow downstream with a 10-0 nylon suture ligating both blood vessels.</li>
		<li>Use micro-dissecting scissors to make a small incision in both the artery and the vein. Insert plastic catheters into both blood vessels. Secure the catheters by tightening the sutures (8-0 or 10-0 nylon sutures dependent on the vessel size) without compromising catheter lumens. Remove the microvascular clamp and close the skin.</li>
		<li>Monitor blood pressure via the arterial catheter. Measure levels of blood gases in arterial blood samples (50 &#956;L) before and after each experiment (normal values: pO2, 90-120 mmHg; pCO2, 35-40 mmHg; pH, 7.25-7.45) using a blood gas analyzer. Use the vein catheter for infusion of fluorescein and anesthesia.</li>
	</ol>
	</li>
	<li>Craniotomy
	<ol>
		<li>Shave the fur off the head of the mouse between the ears. Inject 0.04 mL of 0.5% lidocaine subcutaneously under the planned incision and wait for 2 min.</li>
		<li>Wipe the skull using a 10% iron-chloride solution to remove the periosteum. Rinse the skull thoroughly with saline. Glue a metal head bar to the skull with cyanoacrylate glue and activator (Figure 1A). 
		NOTE: The metal head bar is 75 mm long and 13.5 mm wide, with a round hole of 5 mm diameter in the center.</li>
		<li>Drill a 4-mm-diameter craniotomy, centered at 0.5 mm behind and 3 mm to the right of the bregma over the right sensory barrel cortex. Remove the dura with a fine-tip vessel dilator.</li>
		<li>Cover the exposed cortex with 0.75% agarose gel, dissolved in artificial cerebrospinal fluid (aCSF; pH = 7.4) and cooled to 35 &#176;C. Cover 80-90% of the craniotomy with a glass coverslip at an angle of 10-15&#176; to the metal head bar (Figure 1B) that permits insertion and placement of the glass micro-pipette.</li>
		<li>To minimize brain pulsation, glue the two corners of the glass coverslip onto the metal head bar with cyanoacrylate glue and activator. Apply the activator carefully to prevent getting onto the exposed brain. Rinse the glued glass coverslip thoroughly with saline afterwards.</li>
	</ol>
	</li>
	<li>Perform insertion of the whisker pad stimulation probe. Insert a set of custom-made bipolar electrodes (8 mm length and 0.25 mm thickness) percutaneously into the left side of face to stimulate the ramus infraorbitalis of the trigeminal nerve contralateral to the craniotomy. Position the cathode close to the hiatus infraorbitalis, and insert the anode into the masticatory muscles7. Perform the stimulation with an electrical stimulator at an intensity of 1.5 mA for 1 ms in trains of 20 s at 2 Hz.</li>
	<li>Upon completion of surgery, administer a bolus of 0.05 mL fluorescein isothiocyanate-dextran (FITC-dextran, 4% w/v, molecular weight [MW] 50,000) into the femoral vein to label the blood plasma. Discontinue ketamine and switch the anesthesia to continuous intravenous infusion of &#945;-chloralose (17% w/v, final concentration) mixed with FITC-dextran (2% w/v, final concentration) at 0.02 mL/10 g/h. Wait for about half an hour for stabilization of the new anesthetic state. 
	NOTE: Both FITC-dextran and &#945;-chloralose are dissolved in 0.9% saline.</li>
</ol>
3. First two-photon imaging session
<ol>
	<li>Transfer the mouse to the stage of a commercial two-photon microscope (Table of Materials). Under both red fluorescent protein (RFP, Figure 1C) and green fluorescent protein (GFP, Figure 1C) modes of epi-fluorescent illumination, take a picture of the exposed cortex with a 5x objective. Use the RFP picture as a &#39;map&#39;&#160;for insertion of the glass micro-pipette in the next session. 
	NOTE: The GFP &#39;map&#39;&#160;is used to distinguish veins/venules from arteries/arterioles.</li>
	<li>Perform two-photon imaging using the two-photon microscope and a 25x 1.0 numerical aperture (NA) water-immersion objective with piezo motor. Set the excitation wavelength to 900 nm. Search the cortex and follow each penetrating arteriole and find its horizontal branches (1st order capillaries).</li>
	<li>Image penetrating arterioles and their 1st order capillaries at rest and during whisker pad stimulation. See the detailed imaging parameters in section 5.</li>
	<li>Mark locations of 1st order capillaries with &#62;5% vasodilation during whisker pad stimulation on the RFP &#39;map&#39;&#160;(Figure 1C). Locations with &#60;5% vasodilation are considered as being outside of the whisker barrel cortex region. 
	NOTE: If using wild-type mice instead of NG2DsRed mice in the experiment, the GFP image is used as &#8216;map&#8217; instead.</li>
</ol>
4. Insertion of the glass micro-pipette
<ol>
	<li>Prepare glass micro-pipettes for puffing using a pipette puller (Table of Materials). The glass micro-pipettes have a resistance of 3-3.5 M&#8486; and a taper length of 4.5-5 mm. Load a pipette with a mixture of 1 mM ATP and 10 &#181;M Alexa 594 in order to visualize the pipette tip under the epi-fluorescent lamp and two-photon microscope.</li>
	<li>Mount the glass micro-pipette onto a patch clamp holder and connect it to an air pump. Set the pump holding pressure to 0 psi.</li>
	<li>Using red epi-fluorescent illumination, focus on the pipette tip with the 5x objective. Place the glass micro-pipette into the aCSF above the agarose. Adjust the holding pressure of the air pump to 0.2 psi. A small red cloud can be observed ejecting out of pipette tip in the aCSF. This is to prevent clogging during pipette insertion.</li>
	<li>Choose one of the marked locations in the RFP &#39;map&#39;&#160;as destination. Move the pipette tip to the horizontal plane of the cover glass edge with 30 &#181;m distance, roughly pointing towards the destination in the x-y plane.</li>
	<li>Carefully lower the pipette tip in z-axis under the cover glass slip. Then start advancing the glass pipette towards the destination until ~500 &#181;m above the brain surface. Switch focus frequently between the cover glass edge, the pipette tip and the brain surface, making sure there is enough free space for moving the pipette.</li>
	<li>Switch the objective to 25x. Re-center the pipette tip and advance the pipette tip further towards the destination on the RFP &#39;map&#39;. Keep the pipette tip in the agarose layer.</li>
	<li>When the pipette tip is ~100 &#181;m above the brain surface, switch the imaging mode to two-photon microscopy. Focus on a target location at which to puff. Write down its x, y coordinates (x0, y0) and its depth below the surface (z0). Calculate the coordinates of the insertion point on the brain surface (xi, yi) as follows: 
	xi = x0 + z0 / tan &#952; 
	yi = y0 
	where &#952; is the angle between the pipette holder and the horizontal plane.</li>
	<li>Position the pipette tip to the coordinates (xi, yi) on the brain surface. Gently and slowly insert the pipette until it reaches (x0, y0, z0). If a vessel is in the way of an insertion path, withdraw the pipette and re-calculate other insertion coordinates and path; or turn the stage plate slightly (as indicated in Figure 1A).</li>
	<li>Set the pump holding pressure at 0 psi and ejection pressure at 10-15 psi. Puff ATP for 200-400 ms at the target location during two-photon imaging. Adjust the pressure and duration of puffing for each pipette and over time as explained in the discussion section.</li>
</ol>
5. Hyperstack two-photon imaging 
<ol>
	<li>Perform experiments using the two-photon microscope and a 25 x 1.0 NA water-immersion objective with piezo motor. Set the excitation wavelength to 900 nm. Filter the emitted light to collect red light from DsRed (pericytes)/tetramethylrhodamine isothiocyanate-dextran (TRITC-dextran) staining blood plasma and green light from FITC-dextran (blood plasma)/GCaMP6 (GFP, astrocytic calcium).</li>
	<li>Produce a high-resolution z-stack at the location of interest containing a penetrating arteriole, a 1st order capillary, a 2nd order capillary and possibly neighboring fluorescent cells (e.g., pericytes, astrocytes). Determine the x*y*z volume of hyperstack imaging by including the 3D structure of the blood vessels as much as possible. The total height of each stack may vary from 30-50 &#956;m, while the x-y plane roughly covers an area of 60 &#181;m x 40 &#181;m.</li>
	<li>Set the image stack in the imaging software to be comprised of 8-10 planes with an inter-plane distance of 4-5 &#181;m. The piezo-motor objective stops at each level on the z-axis to acquire the image of each plane. The sampling rate is 1 s per stack and pixel resolution in the x-y plane is 0.2-0.3 &#181;m (Figure 2A). One recording normally includes 10 pre-puff image stacks and 150 post-puff image stacks.</li>
</ol>
6. Data processing
<ol>
	<li>Process data using custom-made analytical software. Flatten each image stack into one image by maximal intensity projection (Figure 2B). Draw a rectangular region of interest (ROI) with a width of 3 &#181;m perpendicularly across the vessel of interest (Figure 2C).</li>
	<li>To minimize interference from shadows of red blood cells and from minor vibrations of the cortex, average the rectangular ROI by projecting it into one line, which then represents the average profile of the vessel segment. Do this for each maximal intensity image.</li>
	<li>Plot the profile lines as a 2D image with the x-axis representing maximal intensity images in chronological order (Figure 2D, upper panel). Use an active contour algorithm (Chan-Vese segmentation) to find the edges of the vessel8,9 (indicated by red curves; Figure 2D, upper panel).</li>
	<li>Calculate the time course of the diameter changes as the difference between the edges of the blood vessel (vertical distance between the upper and lower red curves in Figure 2D, lower panel). Normalize diameter changes to pre-puffing diameter baseline and plot curves of diameter change over time. Do this for each ROI (Figure 2E).</li>
	<li>The vessel response amplitude is defined as the highest/lowest peak amplitude after puffing. The response latency is defined as the latency of half-max amplitude (Figure 2G). Manually trace the skeletonized vascular structure by placing nodes along the vessels (Figure 2F) and measuring geographical distance between each ROI and the penetrating arteriole. Calculate CVR speed by dividing the distances by latency differences.</li>
</ol>
7. Viral vector injection
<ol>
	<li>In order to simultaneously study astrocytic calcium responses, inject a volume of 0.6 &#181;L viral vector (pZac2.1 gfaABC1D-lck-GCaMP6f, Addgene) into the somatosensory cortex of NG2DsRed mice, following the standard stereotactic viral vector injection procedure10.</li>
	<li>After three weeks, astrocytes in mouse somatosensory cortex express the genetically encoded calcium indicator, GCaMP6f. Follow the aforementioned surgical procedure and two-photon imaging for mice. To distinguish between astrocytes and vessel lumina, TRITC-dextran instead of FITC-dextran is used to stain vessel lumina red.</li>
</ol>

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The authors have nothing to disclose.

One challenge for vascular studies is the precise measurement of vessel diameters. The method we describe here used a motorized piezo objective to make fast and repetitive hyperstack imaging by two-photon microscopy. Firstly, this method allows repeated examinations of the penetrating arteriole, 1st order and 2nd order capillaries without loss of focus and led to the discovery of slowly conducted vascular responses in capillaries in vivo. Secondly, in combination with a viral vector injection technique, it enables us to investigate astrocytic calcium responses in three dimensions, which is necessary for studies of blood flow regulation.
This method is not without limitations. Firstly, a narrow rectangular field of view is defined before the 3D imaging in order to achieve high temporal resolution. This usually limits the imaging to first three orders of capillaries. Secondly, lasers of two-photon microscopes must be perfectly aligned. Laser misalignment will falsely de-center each frame and increase the vessel diameter measurement. Thirdly, the sampling rate of 3D imaging is capable of capturing slow CVRs and slow calcium changes in astrocytes. 1-2 Hz per stack is still not fast enough to study fast CVRs11 or fast calcium signals in astrocytes12,13.
Furthermore, in order to affect vessels of interest locally, we have optimized the procedure of precise insertion of a glass micro-pipette into a specific location of the mouse cerebral cortex. There are several critical steps for successful application of this method. Firstly, the angle of the glass micro-pipette holder must be carefully adjusted. It should allow the micro-pipette to freely maneuver beneath the objective and the glass coverslip above the exposed cortex. Secondly, during insertion of the glass micro-pipette into the agarose layer and the cortex, a small holding pressure is necessary to keep the pipette tip unclogged. However, caution must be exerted not to apply too great a holding pressure so that the compound does not leak before puffing. Thirdly, the puffing of the pipette should be tested in agarose layer above the brain to find the optimal pressure and duration releasing enough puffing compound upon one puff while not introducing an obvious displacement of cells and vessels due to mechanical pressure.
In order to make more precise measurement of CVR speed, volume scanning speed should be improved. There are other newly-developed volume-scanning microscopes, for example, a two-photon microscope with an acoustic optical deflector (AOD) scans as fast as 5 volumes per second with the same spatial resolution and volume size (email communication). Light-sheet microscopes scan even a larger volume size (350 &#181;m x 800 &#181;m x 100 &#181;m) with 10 volumes per second14.
Our mouse preparation also has limitations. Acute mouse surgery may have potential complications. Pain relief medicine and anesthesia may affect the neurovascular responses. Acute neuroinflammation can also be triggered, resulting in microglial activation and leukocytosis in cortical tissues and vessels. Although the tip size of glass micro-pipettes is usually very small (&#60;1 &#181;m), the insertion procedure and ATP puffing could also potentially trigger microglial migration and embracement of insertion site within 0.5-1 hour post-insertion.
In conclusion, combination of 3D imaging in two-photon microscopy and localized puffing glass micro-pipette is an advanced tool to study neurovascular activities and their mechanism.

The brain has a high energy consumption rate. About 20% of the oxygen and 25% of the glucose consumed by the human body are dedicated to brain function, while the brain only occupies 2% of the total body mass. Maintenance of normal brain function requires a sufficient and efficient supply of oxygen and nutrition by blood flow in a complex network of vessels. Local brain activity and cerebral blood flow (CBF) are robustly coupled, depending on the functional properties of neurons, astrocytes, pericytes, smooth muscle cells (SMCs) and endothelial cells (ECs)1. Recently, the first few orders of capillaries branching from penetrating arterioles have emerged as a &#39;hotspot&#39;2, showing active regulation of capillary blood flow. A slow conducted vascular response (CVR) was discovered at this &#39;hotspot&#39;&#160;in mouse somatosensory cortex during both whisker stimulation and local ejection (puffing) of ATP with a glass micro-pipette3.
Although in vivo imaging by two-photon laser scanning fluorescent microscopy has been widely used for studying neurovascular responses in cerebral cortex, most of the studies measured blood vessel diameters and investigated their regulation in a two-dimensional (2D) x-y plane. The challenges are: Firstly, cerebral blood vessels and their embracing astrocytes, pericytes and SMCs construct branches in three dimensions (3D). It is therefore crucial to study their interactions in 3D. Secondly, even a small amount of drift in focus will affect the precise measurement of both vessel diameters and cellular fluorescent signals. Finally, CVRs are fast and far-reaching in three dimensions. 3D volume scanning is optimal for detecting CVRs and unveiling their mechanisms. We implemented a piezo motor objective in a two-photon microscope to study mouse somatosensory cortex in vivo, allowing precise diameter measurements by maximal intensity projections of the obtained images.
Glass micro-pipettes have frequently been used for in vivo brain studies, e.g., to bulk-load organic dyes4, record EEGs5 and for patch clamping6. Nonetheless, limitations remain. Commonly, the tip of the glass micro-pipette is imprecisely placed, or the micro-pipette is not used for local interventions. Here, we have optimized the procedure of micro-pipette insertion and local ejection.
Furthermore, the combination of 3D two-photon microscopy and genetically-encoded fluorescent indicators offers an unprecedented opportunity to investigate neurovascular coupling in a 3D scope. In this study, we took advantage of this and injected viral vectors carrying astrocyte specific genetically-encoded calcium indicators into the mouse somatosensory cortex. Astrocytes as well as vessel diameters were imaged simultaneously by combining different fluorescent markers.
Overall, we present an optimized method of local ejection (puffing) by glass micro-pipette and fast two-photon hyperstack imaging, which allows precise measurement of capillary diameter changes. In addition, our method provides a novel tool to simultaneously study 3D profiles of Ca2+ responses in astrocytes and vascular diameter responses.

<table><tbody><tr><td>Agarose</td><td>Sigma&amp;ndash;Aldrich</td><td>A6138</td><td>Apply upon exposed cortex for protection</td></tr><tr><td>Alexa 594</td><td>Life Technologies</td><td>A-10438</td><td>Stain puffing compound to red fluorescent color</td></tr><tr><td>ATP</td><td>Sigma-Aldrich</td><td>A9187</td><td>Vasodilator and vasoconstrictor, puffing compound</td></tr><tr><td>Cyanoacrylate glue and activator</td><td>Loctite</td><td>Adhesives and SF7452</td><td>Glue for metal piece and coverglass</td></tr><tr><td>Eye lubricant</td><td>Neutral Ophtha, Ophtha A/S, Denmark</td><td></td><td>Keep the mouse eyes moisterized</td></tr><tr><td>FITC-dextran</td><td>Sigma-Aldrich</td><td>FD500S</td><td>Blood serum dye, green fluorescent color</td></tr><tr><td>NG2DsRed mice</td><td>Jackson Laboratory</td><td>8241</td><td>These transgenic mice express an red fluorescent protein variant (DsRed) under the control of the mouse NG2 (Cspg4) promoter</td></tr><tr><td>pZac2.1 gfaABC1D-lck-GCaMP6f</td><td>Addgene</td><td>52924-AAV5</td><td>Astrocyte specific viral vectors carrying genetically encoded calcium indicators</td></tr><tr><td>TRITC-dextran</td><td>Sigma-Aldrich</td><td>52194</td><td>Blood serum dye, red fluorescent color</td></tr><tr><td>&lt;strong&gt;List of Equipments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Air pump</td><td>WPI</td><td>PV830</td><td>Give air pressure to pipette puffing</td></tr><tr><td>Blood gas analyzer</td><td>Radiometer</td><td>ABL 700</td><td>Measure levels of blood gases&amp;nbsp;</td></tr><tr><td>Blood pressure monitor</td><td>World Precision Instruments</td><td>BP-1</td><td>Monitor aterial blood pressure</td></tr><tr><td>Body temperature controller</td><td>CWE</td><td>Model TC-1000</td><td>Keep the mouse body temperature in physiological range</td></tr><tr><td>Capnograph</td><td>Harvard Apparatus</td><td>Type 340</td><td>Monitor the end-expiratory CO&lt;sub&gt;2&lt;/sub&gt; from the mouse</td></tr><tr><td>Electrical stimulator</td><td>A.M.P.I.</td><td>ISO-flex</td><td>Apply whisker pad stimulation</td></tr><tr><td>Mechanical ventilator</td><td>Harvard Apparatus</td><td>D-79232</td><td>Mechanically ventilate the mouse in physiological range</td></tr><tr><td>Micropipette puller</td><td>Sutter Instrument</td><td>P-97</td><td></td></tr><tr><td>Two-photon microscope</td><td>Femtonics Ltd</td><td>Femto3D-RC</td><td></td></tr><tr><td>&lt;strong&gt;List of Surgical Instruments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anatomical tweezer&amp;nbsp;</td><td>Lawton</td><td>09-0007</td><td></td></tr><tr><td>Angled and balanced tweezer</td><td>S&amp;amp;T AG</td><td>00595 FRAS-18 RM-8</td><td></td></tr><tr><td>Iris scissor</td><td>Lawton</td><td>05-1450</td><td></td></tr><tr><td>Micro vascular clamp</td><td>S&amp;amp;T AG</td><td>462</td><td></td></tr><tr><td>Mouse vascular catheters</td><td>Verutech</td><td>100828</td><td></td></tr></tbody></table>

in vivo three-dimensional two-photon microscopy to study conducted vascular responses by local atp ejection using a glass micro-pipette

Maintenance of normal brain function requires a sufficient and efficient supply of oxygen and nutrition by a complex network of vessels. However, the regulation of cerebral blood flow (CBF) is incompletely understood, especially at the capillary level. Two-photon microscopy is a powerful tool widely used to study CBF and its regulation. Currently, this field is limited by the lack of in vivo two-photon microscopy studies examining (1) CBF responses in three-dimensions, (2) conducted vascular responses, and (3) localized interventions within the vascular network. Here, we describe a 3D in vivo method using two-photon microscopy to study conducted vascular responses elicited by&#160;local ejection of ATP with a glass micro-pipette. Our method uses fast and repetitive hyperstack two-photon imaging providing precise diameter measurements by maximal intensity projection of the obtained images. Furthermore, we show that this method can also be used to study 3D astrocytic calcium responses. We also discuss the advantages and limitations of glass micro-pipette insertion and two-photon hyperstack imaging.

Once the surgery was complete, mice were transported to two-photon microscope (Figure 1A). A glass micro-pipette containing 1 mM ATP was inserted in proximity of the destination blood vessel at the target location (Figure 1B).
We performed hyperstack imaging while giving a puff of 1 mM ATP (Figure 2A, Supplementary Video 1). Each image stack was flattened to one image by maximal intensity projection (Figure 2B). Rectangular ROIs were placed perpendicularly across the vessel to measure vessel diameter change upon ATP puffing (Figure 2C). Vessel diameters were measured using Chan-Vese segmentation (Figure 2D). In single puff recordings, normalized diameter changes at each ROI were overlaid to compare the responses of different vessel segments (Figure 2E). The distance from each ROI to the penetrating arteriole was calculated by hand-drawing the vascular skeleton (Figure 2F). Amplitudes and latencies of dilation and constriction at each ROI in single puff recordings were plotted over the calculated distances from the ROIs to the penetrating arteriole (Figure 2H-K). Vasodilation upon puffing propagated linearly with a speed of 14.69 µm/s (upstream) and of 2.8 µm/s (downstream), starting from the junction of 1st and 2nd order capillaries (Figure 2H). Vasoconstriction also propagated linearly at 3.92 µm/s, starting from the penetrating arteriole (Figure 2I). Maximal amplitude of both vasodilation and vasoconstriction occurred in 1st order capillaries (Figure 2J-K).
Furthermore, combining astrocyte-specific viral vector-carrying fluorescent calcium indicators and two-photon hyperstack imaging, astrocytic calcium responses to ATP puffing were investigated (Figure 3A, Supplementary Video 2). Rectangular ROIs were placed at astrocytic somata and processes. ATP induced a rise in intracellular calcium in astrocytic processes but not in astrocyte somata (Figure 3B).
<img alt="Rat neuroimaging setup with blood monitoring, microscopy diagram, RFP/GFP maps for vascular study." class="xfigimg" src="/files/ftp_upload/59286/59286fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Diagram of in vivo two-photon 3D imaging and glass micropipette puffing. (A) The anesthetized mouse is head-fixed to a metal bar and mechanically ventilated. End-tidal CO2 is monitored by a capnograph. Both whisker pad stimulation and micro-pipette puffing are used to induce vascular diameter changes. The glass micro-pipette holder is mounted on the stage plate. The femoral artery and vein are catheterized to monitor blood pressure and blood gases, and to infuse anesthesia and fluorescein, respectively. (B) The glass coverslip covers the craniotomy at an angle, which allows free movement of the pipette. The puffing micro-pipette is placed in proximity of a penetrating arteriole and its capillaries and contains a mixture of 10 µM Alexa 594 (red color in glass micro-pipette) and 1 mM ATP. The two-photon imaging is a fast and repetitive 3D volume scanning that includes the penetrating arteriole and the first few order capillaries, as well as neighboring astrocytes and pericytes. (C) Representative images of red fluorescent protein (RFP) and green fluorescent protein (GFP) using epi-fluorescent illumination are shown. They are used as ‘maps’ during pipette insertion. Red circles mark locations of 1st order capillaries with &gt;5% vasodilation during whisker pad stimulation. Scale bar: 50 µm. <a href="https://www.jove.com/files/ftp_upload/59286/59286fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Capillary blood flow dynamics; E: Graphs; C, F: Microscopy images; D: Diameter analysis." class="xfigimg" src="/files/ftp_upload/59286/59286fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: ATP puffing by micro-pipette induces vessel dilation, followed by constriction. (A) Cascade of planes in one representative image stack including penetrating arteriole and 1st and 2nd order capillaries. Pericytes are labeled with a red fluorophore (NG2-DsRed) and the vessel lumen is labeled with FITC-dextran (green). (B) Maximal intensity projection of the image stack. (C) Multiple uniquely colored regions of interest (ROIs) placed perpendicularly across the vasculature to measure the vessel diameter. (D) Top, representative fluorescent intensity over time at the dark blue ROI from panel C. The two red curves define the edges of the vessel wall. The vertical distance between the two red curves is the vessel diameter and is shown as a function of time (bottom). (E) Normalized diameter changes over time at each ROI in response to 1 mM ATP puffing. Measurements are based on a single experiment. (F) The vascular skeleton was manually traced by placing nodes along the vessels. (G) Amplitudes of dilation or constriction were defined as maximal positive or negative vascular response, respectively, during the recording session. The latency of dilations and constrictions were reported as time to half positive or negative maximum after puffing onset. (H-K) Graphs show the latency of dilation (H), the latency of constriction (I), the amplitude of dilation (J), and the amplitude of constriction (K) of all the ROIs shown in panel C versus the distance of each ROI from the penetrating arteriole. The dashed lines represent the linear fitting of upstream and downstream conductive responses. p.a.: penetrating arteriole. Scale bar: 10 µm. <a href="https://www.jove.com/files/ftp_upload/59286/59286fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Fluorescence microscopy with puffing pipette in neuron study; calcium signaling graph data analysis." class="xfigimg" src="/files/ftp_upload/59286/59286fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: ATP puffing by micro-pipette induces astrocytic calcium activities. (A) Astrocytic viral vectors carrying a fluorescent calcium indicator were injected in mouse somatosensory cortex three weeks prior to the experimental procedure. Image shows vessel lumina labeled with TRITC-dextran (red) and astrocytic calcium in green. (B) Multiple ROIs are placed at astrocytic somata and processes. Upon 1 mM ATP puffing, intracellular calcium increased in astrocytic processes but not in somata (dotted boxes), where calcium levels larger than the mean plus 1.5 x standard deviation were defined as significant. Scale bar: 10 µm. <a href="https://www.jove.com/files/ftp_upload/59286/59286fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Fluorescence microscopy; cell dynamics visualization; green-red emission; cellular structure study." src="/files/ftp_upload/59286/59286movie1.jpg" data-altupdatedat="1747911167000" data-alt="Supplementary Video 1"> 
Supplementary Video 1: Time-series movie flattened from hyperstack imaging of vessels in response to puffing of 1 mM ATP. Green: FITC-dextran, staining vessel lumen. Red: NG2DsRed, staining pericytes. <a href="https://www.jove.com/files/ftp_upload/59286/Supplementary_video_1_slow.avi" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Fluorescence microscopy image showing neuron activity with time-lapse, highlighting dendrite tracing." src="/files/ftp_upload/59286/59286movie2.jpg" data-altupdatedat="1747911167000" data-alt="Supplementary Video 2"> 
Supplementary Video 2: Time-series movie flattened from hyperstack imaging of astrocytic calcium in response to puffing of 1 mM ATP. Green: astrocytic calcium. Red: TRITC-dextran, staining vessel lumen. <a href="https://www.jove.com/files/ftp_upload/59286/Supplementary_video_2_slow.avi" target="_blank">Please click here to view this video. (Right-click to download.)</a>

Watch this Scientific Journal Video about In Vivo Three-Dimensional Two-Photon Microscopy to Study Conducted Vascular Responses by Local ATP Ejection Using a Glass Micro-Pipette at JoVE.com

In Vivo Three-Dimensional Two-Photon Microscopy to Study Conducted Vascular Responses by Local ATP Ejection Using a Glass Micro-Pipette

We present an optimized local ejection procedure using a glass micro-pipette and a fast two-photon hyperstack imaging method, which allows precise measurement of capillary diameter changes and investigation of its regulation in three dimensions.

Maintenance of normal brain function requires a sufficient and efficient supply of oxygen and nutrition by a complex network of vessels. However, the ...

in-vivo-three-dimensional-two-photon-microscopy-to-study-conducted

Nanyang Technological University

Research

JoVE Journal

Neuroscience

7.4K Views.  University of Copenhagen. Dit protocol is ontworpen om de langzaam uitgevoerde vasculaire reacties in cerebrale haarvaten veroorzaakt door lokale uitwerping van ATP te bestuderen. Het voordeel van deze techniek is om lokale synaptische activiteiten te simuleren door lokale pipet-uitwerping en om het karige vat scherp te houden door 3D twee-foton microscopie. Deze methode kan ook worden gebruikt voor het bestuderen van cerebrale neuroma en gliaactiviteit in rust en bij stimulatie in drie dimensies. Het aantonen van de ...

Video: In Vivo Three-Dimensional Two-Photon Microscopy to Study Conducted Vascular Responses by Local ATP Ejection Using a Glass Micro-Pipette

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite fluorescent in situ elements through the optical fibers, and then record some of the emitted photons, via the same optical fibers. The light source is a laser that sends the exciting light through an element within the fiber bundle and as it scans over the sample, recreates an image pixel by pixel. As this scan is very fast, by combining it with dedicated image processing software, images in real time with a frequency of 12 frames/sec can be obtained.
We developed a technique to quantitatively characterize capillary morphology and function, using a confocal fluorescence videomicroscopy device. The first step in our experiment was to record 5 sec movies in the four quadrants of the tumor to visualize the capillary network. All movies were processed using software (ImageCell, Mauna Kea Technology, Paris France) that performs an automated segmentation of vessels around a chosen diameter (10 &mu;m in our case). Thus, we could quantify the 'functional capillary density', which is the ratio between the total vessel area and the total area of the image. This parameter was a surrogate marker for microvascular density, usually measured using pathology tools.
The second step was to record movies of the tumor over 20 min to quantify leakage of the macromolecular contrast agent through the capillary wall into the interstitium. By measuring the ratio of signal intensity in the interstitium over that in the vessels, an 'index leakage' was obtained, acting as a surrogate marker for capillary permeability.

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

In this paper, we present a method to analyze tumor microvessels in vivo using dynamic contrast-enhanced fluorescence videomicroscopy. Two quantitative parameters were acquired: functional capillary density reflecting the vascularity of the tumor, and index leakage reflecting the leakiness of the endothelial wall.

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite ...

Medicine

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

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 ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located in. Although traditional imaging techniques are useful in identifying protein interactions and morphology in fixed tissue, they are not able to give insight as to how quickly cells are able to bind and uptake proteins, and once activated how their morphology changes in vivo. In the present study, we ask 2 major questions: 1) what is the time-course of fibroblast activation via toll-like receptor-4 (TLR4) and lipopolysaccharide (LPS) interaction and 2) how do these cells behave once activated? Using 2-photon imaging, we have developed a novel technique to assess the ability of LPS-FITC to bind to its cognate receptor, TLR4, expressed on peripheral fibroblasts in the genetic reporter mouse line; FSP1cre; tdTomatolox-stop-lox&#160;in vivo. This unique approach allows researchers to create in-depth, time-lapse videos and/or pictures of proteins interacting with live cells that allows one to have an increased level of granularity in understanding how proteins can alter cellular behavior.

Morphological changes occur in immune responsive fibroblast cells following activation and promote alterations in cellular recruitment. Utilizing 2-photon imaging in conjunction with a genetically engineered Fibroblast-specific protein 1 (FSP1)-cre; tdTomato floxed-stop-floxed (TB/TB) mouse line and green fluorescently tagged lipopolysaccharide-FITC, we can illustrate highly specific uptake of lipopolysaccharide in dermal fibroblasts and morphological changes in vivo.

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located ...

Bioengineering

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static ...

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

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues throughout the acute injury phase. In fact, endothelial cells appear to be the first to die after a contusive SCI. The early vascular events, including increased permeability of the blood-spinal cord barrier (BSCB), induce vasogenic edema and contribute to detrimental secondary injury events caused by complex injury mechanisms. Targeting the vascular disruption, therefore, could be a key strategy to reduce secondary injury cascades that contribute to histological and functional impairments after SCI. Previous studies were mostly performed on postmortem samples and were unable to capture the dynamic changes of the vascular network. In this study, we have developed an in vivo duo-color two-photon imaging method to monitor acute vascular dynamic changes following contusive SCI. This approach allows detecting blood flow, vessel diameter, and other vascular pathologies at various sites of the same rat pre- and post-injury. Overall, this method provides an excellent venue for investigating vascular dynamics.

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

We introduce an in vivo imaging method using two different fluorescent dyes to track dynamic spinal vascular changes following a contusive spinal cord injury in adult Sprague-Dawley rats.

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues ...

Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of homeostasis, undergo rapid morphological changes. For example, a laser lesion induces in a few minutes an oriented growth of microglial processes, also called &#34;directional motility&#34;, toward the site of injury. A similar effect can be obtained by delivering locally ATP or serotonin (5-hydroxytryptamine [5-HT]). In this article, we describe a protocol to induce a directional growth of microglial processes toward a local application of ATP or 5-HT in acute brain slices of young and adult mice and to image this attraction over time by multiphoton microscopy. A simple method of quantification with free and open-source image analysis software is proposed. A challenge that still characterizes acute brain slices is the limited time, decreasing with age, during which the cells remain in a physiological state. This protocol, thus, highlights some technical improvements (medium, air-liquid interface chamber, imaging chamber with a double perfusion) aimed at optimizing the viability of microglial cells over several hours, especially in slices from adult mice.

Microglia, the resident immune cells of the brain, respond quickly with morphological changes to modifications of their environment. This protocol describes how to use two-photon microscopy to study the attraction of microglial processes toward serotonin or ATP in acute brain slices of mice.

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

Biology

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for analyzing collateral arteries and leukocytes in arteriogenesis is ex vivo (immuno-) histological methodology. However, this technique does not allow the measurement of dynamic processes such as blood flow, shear stress, cell-cell interactions, and particle velocity. This paper presents a protocol to monitor in vivo processes in growing collateral arteries during arteriogenesis utilizing intravital imaging. The method described here is a reliable tool for dynamics measurement and offers a high-contrast analysis with minimal photo-cytotoxicity, provided by multiphoton excitation microscopy. Prior to analyzing growing collateral arteries, arteriogenesis was induced in the adductor muscle of mice by unilateral ligation of the femoral artery.
After the ligation, the preexisting collateral arteries started to grow due to increased shear stress. Twenty-four hours after surgery, the skin and subcutaneous fat above the collateral arteries were removed, constructing a pocket for further analyses. To visualize blood flow and immune cells during in vivo imaging, CD41-fluorescein isothiocyanate (FITC) (platelets) and CD45-phycoerythrin (PE) (leukocytes) antibodies were injected intravenously (i.v.) via a catheter placed in the tail vein of a mouse. This article introduces intravital multiphoton imaging as an alternative or in vivo complementation to the commonly used static ex vivo (immuno-) histological analyses to study processes relevant for arteriogenesis. In summary, this paper describes a novel and dynamic in vivo method to investigate immune cell trafficking, blood flow, and shear stress in a hindlimb model of arteriogenesis, which enhances evaluation possibilities notably.

The recruitment of leukocytes and platelets constitutes an essential component necessary for the effective growth of collateral arteries during arteriogenesis. Multiphoton microscopy is an efficient tool for tracking cell dynamics with high spatio-temporal resolution in vivo and less photo-toxicity to study leukocyte recruitment and extravasation during arteriogenesis.

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for ...

In Vivo Three-Dimensional Two-Photon Microscopy to Study Conducted Vascular Responses by Local ATP Ejection Using a Glass Micro-Pipette

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