The study was funded by the Deutsche Forschungsgemeinschaft SFB 914 (HI-A/SM, project Z01). We thank Dr. Susanne Stutte for reading the manuscript.

This study was approved by the Bavarian Animal Care and Use Committee (approved by ethical code: ROB-55.2Vet-2532.Vet_02-17-99); these experiments were carried out in strict accordance with the German animal legislation guidelines.
1. Animals and femoral artery ligation (FAL)
NOTE: To induce sterile inflammation and arteriogenesis, 8-10 weeks old male C57BL/6J mice were used. None of the mice died or suffered from wound infection or wound healing disturbance during or post-FAL or sham surgery, respectively.
<ol>
	<li>Anesthesia 
	NOTE: Before injection of the MMF-mix anesthesia, it is recommended to first anesthetize with 5% isoflurane until the mouse is sleeping.
	<ol>
		<li>Weigh the mouse and prepare MMF mix with a dose of midazolam 5.0 mg/kg, medetomidine 0.5 mg/kg, and fentanyl 0.05 mg/kg.</li>
		<li>Fill a 1 mL syringe with MMF-mix and inject a final volume of 100 &#181;L s.c. using a 30 G needle (after anesthesia with 5% isoflurane).</li>
		<li>Place the mouse on a warm pad and wait until the mouse is completely anesthetized. Set the timer to 35 min. After this time, inject s.c. half of the dose (50 &#181;L) of the MMF-mix.</li>
		<li>Check the depth of anesthesia by testing the pedal and interdigital reflexes (by firm toe pinch) of the mouse. 
		NOTE: An absence of response indicates an adequate depth of&#160;analgesia.</li>
	</ol>
	</li>
	<li>Femoral artery ligation (FAL) 
	NOTE: Use sterile surgical instruments and sutures. Disinfect the skin with disinfectant before opening and after closing.
	<ol>
		<li>Place the anesthetized mouse on a heating plate (35-37 &#176;C) to maintain physiological body temperature. Apply eye cream (dexpanthenol) to each eye to prevent drying of the eyes under anesthesia.</li>
		<li>Transfer the anesthetized mouse to a Laser Doppler imaging (LDI) chamber before and after FAL to check blood flow and perfusion (Figure 1A-C).</li>
		<li>Remove hair, disinfect the skin, and make a cut to access the femoral artery. Under a surgical microscope, ligate the right femoral artery of the mouse hind limb using a braided silk suture (7-0) and perform a sham operation on the left femoral artery to serve as an internal control as previously described6 (Figure 1D-K). 
		NOTE: The incision in the skin was made with small scissors to avoid direct injury to the blood&#160;vessels close to the skin.</li>
		<li>Close the skin using a braided silk suture (6-0). Administer buprenorphine (0.1 mg/kg) s.c. every 12 h, starting 10 min before antagonizing the anesthesia for pain relief. Observe the animal for signals of pain, i.e., reduced grooming activity, reluctance to move, or hunched posture for 24 h post-surgery.</li>
		<li>Inject (s.c.) a combination of naloxone (1.2 mg/kg), flumazenile (0.5 mg/kg), and atipamezole (2.5 mg/kg) to antagonize the anesthesia after finishing the FAL and closing the skin.</li>
		<li>After surgery, keep the mouse on the warm pad and continuously monitor the animal until it can maintain an upright posture and start walking normally around the cage.</li>
		<li>When the mouse is fully awake, place it back in the cage together with the other mice and return the cage to the animal housing room.</li>
	</ol>
	</li>
</ol>
2. Tissue preparation for multiphoton intravital imaging
<ol>
	<li>Tail vein injection 
	NOTE: The animal needs to be under anesthesia before starting the preparation for intravital imaging. Before introducing the needle of the vein catheter, it is recommended to apply a skin disinfectant on the tail. This will also help to visualize the vessels.
	<ol>
		<li>Prepare a tail vein injection catheter using a 2 x 30 G needle, 10 cm of a fine bore polythene tubing (0.28 mm internal diameter and 0.61 mm outer diameter), a 1 mL syringe, and a histoacryl glue to fix the catheter on the mouse tail for further i.v. injections during imaging (Figure 2A,B and Figure 2F).</li>
		<li>Prepare the antibody solution for i.v. injection: CD45-PE and CD41-FITC (20 &#181;g/mouse) at a final volume of 100 &#181;L.</li>
		<li>Check the tail for the laterally located tail veins and dam the vein to identify the tail vein. Disinfect the tail, insert the vein catheter, and fix it using tissue histoacryl glue (Figure 2B).</li>
		<li>Inject the antibodies prior to imaging (at least 15 min before) and after finishing the tissue preparation.</li>
	</ol>
	</li>
	<li>Tissue preparation 
	NOTE: Always use a warm pad and administer a drop of eye cream (dexpanthenol) in each eye before tissue preparation. Use sterile surgical instruments and sutures. Disinfect the skin with disinfectant before open and after closing.
	<ol>
		<li>Place the anesthetized mouse on a stable and portable pad. Fix the mouse in a supine position with a 4 point fixation on the pad using adhesive tape (Figure 2B).</li>
		<li>Mold two pieces of modeling clay and place the pieces under the upper hind limb of the mouse to ensure a plane position of the adductor muscle (Figure 2B, indicated by red arrows).</li>
		<li>To ensure a stable body temperature of 37 &#176;C of the mouse, place the pad with the mouse on a heating plate under a surgical microscope with a 0.64-4.0-fold zoom (Figure 1L).</li>
		<li>Remove the hair from both legs, disinfect the skin, and cut the skin in a circle around the scar of the earlier femoral artery ligation of the right hind limb (Figure 1M).</li>
		<li>Remove the skin and fat layer from the top of the leg (Figure 1N,O), and pull aside the remaining skin using sutures to create a pocket around the adductor muscle (Figure 1P,Q).</li>
		<li>Remove subcutaneous fat to get a clear vision of the adductor muscle and the profound artery/vein as well as the collateral vessels (Figure 1Q,R).</li>
		<li>Carefully start removing the superficial muscle layer on top of the collateral vessels by carefully dissecting it out with fine forceps (Figure 1R). 
		NOTE: The collateral artery and the collateral vein are clearly visible and ready for imaging (Figure 1S).</li>
		<li>Fill the prepared pocket with saline to avoid drying of the tissue and continue with the same procedure for the sham-operated left hind limb. Before imaging, add ultrasonic gel in both pockets, which prevents drying and serves as an immersion medium for optical coupling with the objective (Figure 2C and Figure 2F).</li>
	</ol>
	</li>
</ol>
3. Intravital multiphoton microscopy
<ol>
	<li>Preparation 
	NOTE: Keep a syringe with ultrasonic gel inside the incubator chamber of the microscope to maintain a warm temperature for refilling the pockets during long-term imaging.
	<ol>
		<li>Remove the saline from the two prepared pockets and replace it with ultrasonic gel to cover the collateral vessels (Figure 2C).</li>
		<li>Inject the antibody solution through the tail vein catheter.&#160;15 min after antibody injection, transfer the pad with the fixed mouse into the warmed multiphoton imaging chamber (Figure 2F).</li>
		<li>After completion of the image acquisition, euthanize the mouse by cervical dislocation under deep narcosis.</li>
	</ol>
	</li>
	<li>Multiphoton microscopy image acquisition 
	NOTE: Turn on the microscope 30 min before imaging to allow for laser stabilization. For the best optical stability of the microscope, it is recommended to keep the microscope chamber permanently heated.
	<ol>
		<li>Turn on the key of the Ti:Sapphire laser box, the electronic interfaces boxes, the heating unit of the incubation chamber, the fluorescent lamp, and the computer (Figure 2D,E).</li>
		<li>Launch the acquisition software (Inspector software). Set the wavelength to 800 nm and open the microscope shutter (Figure 3A ii).</li>
		<li>In the Measurement Wizard dialog, choose the Instrument Mode (Single beam) and in Measurement Mode (3D-scan Time lapse) (Figure 3A i).</li>
		<li>Transfer the anesthetized mouse into the pre-warmed incubation chamber of the microscope. Position the area with the ultrasonic gel (step 2.2.8) directly in contact with the objective front lens (Figure 2F).</li>
		<li>Open the epifluorescence microscope shutter, choose an adequate filter/dichroic setting for FITC visualization (4) to find the area of interest by following the blood flow under epifluorescence illumination. After bringing the area of interest into the center field of view, close the epifluorescence microscope shutter.</li>
		<li>In the xy-Scanner dialog, set the following parameters: Image size (554x554), Pixel (512x512), Frequency (400), Line average (1) (Figure 3A iii). Adjust the laser power (5%) (Figure 3A iv), and set the photomultiplier (PMT) gain for the channels green (66), red (66), and blue (63) (Figure 3A v).</li>
		<li>Select anadequate objective for intravital imaging and drift correction settings (see details in 3.2.11). 
		NOTE: Here, the selected objective was the Nikon LWD 16x (Figure 3A vi).</li>
		<li>Define the image stack range by starting the preview acquisition mode by pressing the red button on the top right corner of the Measurement Wizard dialog window (Figure 3A i).</li>
		<li>Define the area and structure of interest by focusing to obtain a good image. Then, while observing the screen, change focus by moving the objective up until the image disappears, and set it as zero (first= 0). Change focus in the opposite direction by moving the objective down until the image disappears from the screen again, and set it as the last (end= 40 &#181;m) position in the axial direction (Figure 3A vii).</li>
		<li>Set the step size to 2 &#181;m. Click the red button on the top right corner of the Measurement Wizard dialog to stop the preview scanning. 
		NOTE: The number of images will be calculated and set automatically (21 images), as indicated in Figure 3A vii.</li>
		<li>If the microscope is equipped with live-drift correction9, start the drift correction software (e.g., VivoFollow) and follow the steps described below.
		<ol>
			<li>In the Measurement Wizard dialog, choose the Python axis (Python Ax) (Figure 3A viii) as the first axis device; DO NOT activate the auto save mode. Check the auto save (AS) box only on the time axis (Time Time). Press the Python icon on the Available Devices window (Figure 3A ix) to open the Python dialog window.</li>
			<li>In the Python dialog Axis settings, enter in From (0 zero) and To (-40). Enter Number of steps (21) as indicated in Figure 3A x.</li>
			<li>Go to the xyz Stage Z dialog window and enlarge the Scan Range by 200 &#181;m on both ends: in Start, enter (200 &#181;m) and in End, enter (-240 &#181;m), the range will automatically be set (-440 &#181;m). Keep the step size (2 &#181;m) as shown in Figure 3A xi.</li>
			<li>Open the VivoFollow Frontend dialog and click the Motion Profile box (Figure 3A xii).</li>
			<li>Start image acquisition by pressing the green arrowhead in the top left corner of the Measurement Wizard dialog of the Inspector Software (Figure 3A i).</li>
			<li>If live-drift correction is used, look for a dialog for the drift correction configuration to appear as shown in Figure 3A xiii. Set the acquisition channel that will be used as an immobile landmark reference (for this protocol, choose channel 2, where the vascular tracer signal is to be recorded). Enter the maximal correction offset in &#181;m that was added to the first and last z positions (here, 200 &#181;m) and click OK.</li>
			<li>Monitor the current drift offset in x, y, and z in real time during the image acquisition in the VivoFollow fronted dialog window Figure 3A xiv.</li>
			<li>Stop imaging acquisition after ~35 min when another cycle of anesthesia re-injection is required. Inject half a dose of the MMF-mix (50 &#181;l s.c) and re-start imaging acquisition by pressing the green arrowhead again (step 3.2.11.5).</li>
			<li>Repeat this procedure until the experiment is finished.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol>
	<li>The top 10 causes of death. World Health Organization Available from: <a target="_blank" href="https://www.who.int/news-room/fact-sheets/detail/the-top-10-causes-of-death">https://www.who.int/news-room/fact-sheets/detail/the-top-10-causes-of-death</a> (2020)</li><li>Deindl, E., Schaper, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+art+of+arteriogenesis.">The art of arteriogenesis.</a> Cell Biochemistry and Biophysics. 43 (1), 1-15 (2005).</li><li>Lasch, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+RNA+released+due+to+shear+stress+controls+natural+bypass+growth+by+mediating+mechanotransduction+in+mice.">Extracellular RNA released due to shear stress controls natural bypass growth by mediating mechanotransduction in mice.</a> Blood. 134 (17), 1469-1479 (2019).</li><li>Kumaraswami, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+effective+flow+Ccytometry-based+method+for+identification+and+quantification+of+tissue+infiltrated+leukocyte+subpopulations+in+a+mouse+model+of+peripheral+arterial+disease.">A simple and effective flow Ccytometry-based method for identification and quantification of tissue infiltrated leukocyte subpopulations in a mouse model of peripheral arterial disease.</a> International Journal of Molecular Sciences. 21 (10), 3593 (2020).</li><li>Padgett, M. E., McCord, T. J., McClung, J. M., Kontos, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+acute+and+subacute+murine+hindlimb+ischemia.">Methods for acute and subacute murine hindlimb ischemia.</a> Journal of Visualized Experiments: JoVE. (112), e54166 (2016).</li><li>Limbourg, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+postnatal+arteriogenesis+and+angiogenesis+in+a+mouse+model+of+hind-limb+ischemia.">Evaluation of postnatal arteriogenesis and angiogenesis in a mouse model of hind-limb ischemia.</a> Nature Protocols. 4 (12), 1737 (2009).</li><li>Wagner, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+of+monocyte+homing+and+tumor-related+angiogenesis+in+a+murine+model+of+peripheral+arterial+disease.">Intravital microscopy of monocyte homing and tumor-related angiogenesis in a murine model of peripheral arterial disease.</a> Journal of Visualized Experiments: JoVE. (126), e56290 (2017).</li><li>Ishikawa-Ankerhold, H. C., Ankerhold, R., Drummen, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+fluorescence+microscopy+techniques--FRAP,+FLIP,+FLAP,+FRET+and+FLIM.">Advanced fluorescence microscopy techniques--FRAP, FLIP, FLAP, FRET and FLIM.</a> Molecules. 17 (4), 4047-4132 (2012).</li><li>Vladymyrov, M., Abe, J., Moalli, F., Stein, J. V., Ariga, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+tissue+offset+correction+system+for+intravital+multiphoton+microscopy.">Real-time tissue offset correction system for intravital multiphoton microscopy.</a> Journal of Immunological Methods. 438, 35-41 (2016).</li><li>Chandraratne, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+role+of+platelet+glycoprotein+ibalpha+in+arterial+remodeling.">Critical role of platelet glycoprotein ibalpha in arterial remodeling.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 35 (3), 589-597 (2015).</li></ol>

The authors have no conflicts of interest to declare.

The described method of multiphoton in vivo analysis of leukocyte recruitment represents an addition to commonly used tools for leukocyte recruitment studies such as (immuno-) histological or FACS analyses. With this imaging method, it is possible to visualize in greater detail the dynamic processes in leukocyte adherence and extravasation during arteriogenesis10. Despite the added value of this method, the offered protocol includes some critical steps and limitations. See Table 1...

Despite intensive research interest during recent years, cardiovascular diseases, e.g., ischemic heart disease and stroke, are still the leading global cause of death1. Current treatments for these diseases are highly invasive therapies such as percutaneous transluminal angioplasty, percutaneous transluminal coronary angioplasty, or bypass surgery2. Therefore, the development of alternative, non-invasive therapeutic options is urgently needed. The body can create natural bypasses around a stenosed or occluded vessel to redirect the interrupted blood flow to the distal part of the stenosis. This process is called arteriogenesis2. Many recent studies have shown that increased fluid shear impacts leukocyte recruitment, which plays an important role during arteriogenesis3. The main current options to analyze the recruitment of leukocytes during arteriogenesis are ex vivo (immuno-) histological analyses or fluorescence-activated cell sorting (FACS) methodology4. To enable the assessment of leukocyte dynamics during arteriogenesis, this paper presents an intravital imaging protocol with multiphoton microscopy.
Leukocytes are the major blood cells recruited during the process of arteriogenesis3. This protocol uses multiphoton imaging to show the crawling of adherent leukocytes labeled with injected anti-CD45-PE antibodies in collateral arteries, 24 h after the induction of arteriogenesis by femoral artery ligation (FAL) employing a murine hindlimb ischemia model5,6. Alternatively, immune cells can be labeled ex vivo and carefully injected into mice, as shown in studies on angiogenesis using intravital microscopy7. The blood flow inside vessels and arteries can be visualized by CD41-FITC (to label platelets), dextran-FITC (plasma), or by the second harmonic generation (SHG), which visualizes collagen type 1 present in the basement membrane of some part of the vascular tree. SHG is a unique free labeling effect of the multiphoton excitation. Multiphoton imaging allows long-term cell tracking without harming the tissue and activating the cells by laser power excitation. Multiphoton microscopy is the imaging method of choice for visualizing fluorescently labeled cells and structures in living animals due to its ability to excite the fluorophores deeper into the tissue/organs with minimal phototoxicity8.
The use of tuned infrared lasers with pulses delivered within femtosecond intervals excites the fluorochrome only at the focal plane, with no excitation above and below the focal plane8. Thus, multiphoton microscopy allows high spatio-temporal resolution, less photo-damage, and increased tissue penetration imaging for studying dynamic biological events inside the organs. It is an ideal microscopy tool for live-animal imaging. However, multiphoton and any other art of intravital microscopy is limited by tissue motion due to heartbeat, respiration, peristaltic movements, muscle tonality, and other physiological functions, which disturbs imaging acquisition and analysis. As these movements impair temporal and spatial resolution and sometimes even prohibit subsequent analysis, they must be addressed appropriately to enable accurate data analysis and interpretation. Several strategies have been developed to lower or prevent artifacts from tissue motion. This protocol applies an in situ drift correction software called VivoFollow9 to correct tissue drift during image acquisition. This approach provides the required image stabilization, enabling long-term imaging and cell-tracking analysis.

<table><tbody><tr><td>1.0 mL Syringe</td><td>BD Biosciences, San Jose, CA, USA</td><td>309628</td><td>syringe for injection</td></tr><tr><td>3M Durapore Surgical Tape 1538-0</td><td>3M, St. Paul, MN, USA</td><td>1538-0</td><td>fixation tape</td></tr><tr><td>Atipamezole</td><td>Zoetis, Berlin, Germany</td><td></td><td>antagonize anesthesia</td></tr><tr><td>Buprenorphine</td><td>Reckitt Benckiser Healthcare, Slough, UK</td><td></td><td>antagonize anesthesia</td></tr><tr><td>C57/B6J mouse</td><td>Charles River, Sulzfeld, Germany</td><td></td><td>used animals for surgery/imaging</td></tr><tr><td>CD41-FITC ab</td><td>Biolegend</td><td>133904</td><td>Platelet labeling in vivo</td></tr><tr><td>CD45-PE ab</td><td>Biolegend</td><td>368510</td><td>Leukocytes labelling in vivo</td></tr><tr><td>Disinfectant Cutasept</td><td>Carl Roth GmbH, Karlsruhe, Deutschland</td><td>AK64.2</td><td>Disinfection</td></tr><tr><td>Eye cream (Bepanthen)</td><td>Bayer Vital GmbH</td><td>5g</td><td></td></tr><tr><td>Fentanyl</td><td>CuraMED Pharma, Karlsruhe, Germany</td><td></td><td>anesthesia</td></tr><tr><td>Flumazenile</td><td>Inresa Arzneimittel GmbH, Freiburg, Deutschland</td><td></td><td>antagonize anesthesia</td></tr><tr><td>Fine bore polythene tubing</td><td>Smiths medical</td><td>Lot 278316</td><td>0.28 mm ID and 0.61 mm OD, tubing for the vein catheter</td></tr><tr><td>Histoacryl flexible</td><td>BRAUM</td><td>1050052</td><td>tissue glue</td></tr><tr><td>Imaris software</td><td>Oxford Instruments</td><td>version 9.2</td><td>Used for cell tracking, cell speed analysis, 3D projection</td></tr><tr><td>Laser Doppler Imaging instrument</td><td>Moor LDI 5061 and Moor Software Version 3.01, Moor Instruments, Remagen, Germany</td><td></td><td></td></tr><tr><td>LEICA KL300 LED</td><td>Leica, Solms, Germany</td><td></td><td>light for microscope</td></tr><tr><td>Leica M60</td><td>Leica, Solms, Germany</td><td></td><td>microscope for surgery</td></tr><tr><td>LEICA MC120 HD</td><td>Leica, Solms, Germany</td><td></td><td>camera for microscope</td></tr><tr><td>Medetomidine</td><td>Pfizer Pharma, Berlin, Germany</td><td></td><td>anesthesia</td></tr><tr><td>Midazolam</td><td>Ratiopharm GmbH, Ulm, Germany</td><td></td><td>anesthesia</td></tr><tr><td>Multiphoton microscope</td><td>Lavision</td><td>TRIMScope II</td><td>WL 820 nm</td></tr><tr><td>NaCl 0.9%</td><td>Braun, Melsungen, Deutschland</td><td>3570310</td><td>saline for pocket</td></tr><tr><td>Naloxone</td><td>Inresa Arzneimittel GmbH, Freiburg, Deutschland</td><td></td><td>antagonize anesthesia</td></tr><tr><td>Needle 30 G</td><td>BD Biosciences, San Jose, CA, USA</td><td>305128</td><td>needle for i.v. catheter</td></tr><tr><td>Silk braided suture (7-0)</td><td>Pearsalls Ltd., Taunton, UK</td><td>SUT-S&amp;nbsp;103</td><td>suture for femoral artery ligation</td></tr><tr><td>Ultrason Gel</td><td>SONOSID-ASID BONZ 250 mL</td><td>782012</td><td>gel for imaging</td></tr><tr><td>Vicryl 6.0 suture</td><td>Vicryl, Johnson&amp;amp;Johnson, New Brunswick, NJ, USA</td><td>NW-2347</td><td>suture to build pocket</td></tr><tr><td>VivoFollow drift correction software</td><td>Developed by Mykhailo Vladymyrov</td><td></td><td>Reference 9</td></tr></tbody></table>

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.

Multiphoton microscopy offers a high spatio-temporal resolution for leukocyte tracking, wherein cell migration steps and speed can be tracked and monitored (Figure 4A,B). However, the physiological motion of the sample poses a challenge, especially for long-term intravital microscopy image acquisitions. Therefore, a good tissue preparation and holders to fix the tissue and tools, such as real-time imaging correction for tissue drift, are required for successful and stable im...

Watch this Scientific Journal Video about Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model at JoVE.com

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

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 ...

multiphoton-intravital-imaging-for-monitoring-leukocyte-recruitment

Research

JoVE Journal

Medicine

1.3K Views.  Ludwig-Maximilians-Universität München. Ons protocol stelt onderzoekers in staat om het proces van arteriogenese in vivo te analyseren door de hechting en extravasatie van immuuncellen in groeiende collaterale slagaders in realtime te volgen. De multifotonenmicroscopietechniek maakt de visualisatie mogelijk van celdynamica in het diepe weefsel van levende muismodellen met lage fototoxiciteit en met een hoge spatiotemporale resolutie. De microscoopprocedure wordt gedemonstreerd door Dominic van den Heuvel, een technisch assistent va...

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

Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung is the most difficult to assess using intravital optical imaging due to its enclosed position within the body, delicate nature and vital role in sustaining proper physiology. While clinical modalities (positron emission tomography (PET), magnetic resonance imaging (MRI) and computed tomography (CT)) are capable of providing noninvasive images of this tissue, they lack the resolution necessary to visualize the earliest seeding events, with a single pixel consisting of nearly a thousand cells. Current models of metastatic lung seeding postulate that events just after a tumor cell&#39;s arrival are deterministic for survival and subsequent growth. This means that real-time intravital imaging tools with single cell resolution 2 are required in order to define the phenotypes of the seeding cells and test these models. While high resolution optical imaging of the lung has been performed using various ex vivo preparations, these experiments are typically single time-point assays and are susceptible to artifacts and possible erroneous conclusions due to the dramatically altered environment (temperature, profusion, cytokines, etc.) resulting from removal from the chest cavity and circulatory system 3. Recent work has shown that time-lapse intravital optical imaging of the intact lung is possible using a vacuum stabilized imaging window 2,4,5 however, typical imaging times have been limited to approximately 6 hr. Here we describe a protocol for performing long-term intravital time-lapse imaging of the lung utilizing such a window over a period of 12 hr. The time-lapse image sequences obtained using this method enable visualization and quantitation of cell-cell interactions, membrane dynamics and vascular perfusion in the lung. We further describe an image processing technique that gives an unprecedentedly clear view of the lung microvasculature.

This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window.

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung ...

Cancer Research

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Immunology and Infection

Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the postcapillary venules of inflamed tissue, leukocytes initially tether and roll on the luminal surface of venular wall. Rolling leukocytes arrest on endothelium and undergo firm adhesion in response to chemokine or other chemoattractants on the venular surface. Many adherent leukocytes relocate from the initial site of adhesion to the junctional extravasation site in endothelium, a process termed intraluminal crawling3. Following crawling, leukocytes move across endothelium (transmigration) and migrate in extravascular tissue toward the source of chemoattractant (chemotaxis)4. Intravital microscopy is a powerful tool for visualizing leukocyte-endothelial cell interactions in vivo and revealing cellular and molecular mechanisms of leukocyte recruitment2,5. In this report, we provide a comprehensive description of using brightfield intravital microscopy to visualize and determine the detailed processes of neutrophil recruitment in mouse cremaster muscle in response to the gradient of a neutrophil chemoattractant. To induce neutrophil recruitment, a small piece of agarose gel (~1-mm3 size) containing neutrophil chemoattractant MIP-2 (CXCL2, a CXC chemokine) or WKYMVm (Trp-Lys-Tyr-Val-D-Met, a synthetic analog of bacterial peptide) is placed on the muscle tissue adjacent to the observed postcapillary venule. With time-lapsed video photography and computer software ImageJ, neutrophil intraluminal crawling on endothelium, neutrophil transendothelial migration and the migration and chemotaxis in tissue are visualized and tracked. This protocol allows reliable and quantitative analysis of many neutrophil recruitment parameters such as intraluminal crawling velocity, transmigration time, detachment time, migration velocity, chemotaxis velocity and chemotaxis index in tissue. We demonstrate that using this protocol, these neutrophil recruitment parameters can be stably determined and the single cell locomotion conveniently tracked in vivo.

We describe a protocol of brightfield intravital microscopy for measuring dynamic neutrophil-endothelial cell interactions during neutrophil recruitment in response to the source of a neutrophil chemoattractant in vivo. Neutrophil intraluminal crawling, transendothelial migration and chemotaxis in mouse cremaster muscle tissue are visualized with time-lapsed video photography and tracked with ImageJ.

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the ...

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this protocol, we describe intravital microscopy of mesentery veins. This can be performed in a short period of time with reproducible results showing leukocyte-endothelial interactions in vivo. We describe an inflammatory setting after LPS challenge of the endothelium. But in this model one can apply many different types of inflammatory conditions, like bacterial, chemical or biological and investigate the administration of drugs and their direct effects on the living animal and its impact on leukocyte recruitment. This protocol has been applied successfully to a number of different treatments of mice and their effects on inflammatory response in vessels. Herein, we describe the visualization of leukocytes and platelets by fluorescently labeling these with rhodamine 6G. Additionally, any specific imaging can be performed using targeted fluorescently labeled molecules.

This simplified application of intravital microscopy of mesentery veins in mice can be used in different models of inflammation to observe leukocyte-endothelial and platelet-leukocyte interactions.

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Intravital Imaging of Neutrophil Priming Using IL-1&#946; Promoter-driven DsRed Reporter Mice

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that enhances the phagocytic functionality of neutrophils. Although extensive studies have unveiled the existence and importance of neutrophil priming during infection and injury, means of visualizing this process in vivo have been unavailable. The protocol provided enables monitoring of the dynamic process of neutrophil priming in living animals by combining three methodologies: 1) DsRed reporter signal &#8212; used as a measure of priming 2) in vivo neutrophil labeling &#8212; achieved by injection of fluorescence-conjugated anti-lymphocyte antigen 6G (Ly6G) monoclonal antibody (mAb) and 3) intravital confocal imaging. Several critical steps are involved in this protocol: oxazolone-induced mouse ear skin inflammation, appropriate sedation of animals, repeated injections of anti-Ly6G mAb, and prevention of focus drift during imaging. Although a few limitations have been observed, such as the limit of continuous imaging time (~ 8 hr) in one mouse and the leakage of fluorescein isothiocyanate-dextran from blood vessels in the inflammatory state, this protocol provides a fundamental framework for intravital imaging of primed neutrophil behavior and function, which can easily be expanded to examination of other immune cells in mouse inflammation models.

This current protocol employs fluorescent reporters, in vivo labeling, and intravital imaging techniques to enable monitoring of the dynamic process of neutrophil priming in living animals.

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that ...

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, including hematopoiesis, immune regulation and tumor regulation. Commonly used methods for understanding cellular actions in the bone marrow, such as histology and blood counts, provide static information rather than capturing the dynamic action of multiple cellular components in vivo. To complement the standard methods, a window chamber (WC)-based model was developed to enable serial in vivo imaging of cells and structures in the murine bone marrow. This protocol describes a surgical procedure for installing the WC in the femur, in order to facilitate long-term optical access to the femoral bone marrow. In particular, to demonstrate its experimental utility, this WC approach was used to image and track neutrophils within the vascular network of the femur, thereby providing a novel method to visualize and quantify immune cell trafficking and regulation in the bone marrow. This method can be applied to study various biological processes in the murine bone marrow, such as hematopoiesis, stem cell transplantation, and immune responses in pathological conditions, including cancer.

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

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 ...

Intravital Microscopy of Monocyte Homing and Tumor-Related Angiogenesis in a Murine Model of Peripheral Arterial Disease

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic stenosis. Vascular surgery is a viable option in selected cases, but for patients without indications for surgery such as progression to rest pain, critical limb ischemia, or major disruptions to life or work, there are few possibilities for mitigating their disease. Cell therapy via monocyte-enhanced perfusion through the stimulation of collateral formation is one of a few non-invasive options.
Our group examines arteriogenesis after monocyte transplantation into mice using the hindlimb ischemia model. Previously, we have demonstrated improvement in hindlimb perfusion using tetanus-stimulated syngeneic monocyte transplantation. In addition to the effects on the collateral formation, tumor growth could be affected by this therapy as well. To investigate these effects, we use a basement membrane-like matrix mouse model by injecting the extracellular matrix of the Engelbreth-Holm-Swarm sarcoma into the flank of the mouse, after occlusion of the femoral artery.
After the artificial tumor studies, we use intravital microscopy to study in vivo tumor-angiogenesis and monocyte homing within collateral arteries. Previous studies have described the histological examination of animal models, which presupposes subsequent analysis to post-mortem artifacts. Our approach visualizes monocyte homing to areas of collateralization in real time sequences, is easy to perform, and investigates the process of arteriogenesis and tumor angiogenesis in vivo.

Monocytes are important mediators of arteriogenesis in the context of peripheral arterial disease. Using a basement membrane-like matrix and intravital microscopy, this protocol investigates monocyte homing and tumor-related angiogenesis after monocyte injection in the femoral artery ligation murine model.

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

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