We would like to thank Ying Yu for ApcMin/+ mice genotyping. This study was supported by funds from INSERM and grants (CA057621 and AI053194) from the National Institutes of Health.

NOTE: All animal experiments were conducted in accordance with procedures approved by the Institutional Animal Care and Use Committee (IACUC), UCSF. All the imaging experiments were non-survival procedures and the animals were euthanized immediately following the end of image acquisition.
1. Generation of Mice
NOTE: ApcMin/+ mice, carrying a mutation on the Apc gene, spontaneously develop 50-100 adenomas in the small intestine.
<ol>
	<li>Cross ApcMin/+ mice with the ACTB-ECFP line, which expresses ECFP under the actin promoter, and further with the c-fms-EGFP line, which expresses EGFP under the c-fms promoter to detect the myeloid cells. Heterozygous animals for ACTB-ECFP and c-fms-EGFP are used to detect fluorescence.</li>
	<li>Use mice at 3-4 months of age to image clearly detectable adenomas.</li>
</ol>
2. Preparation of Materials Before Surgery
NOTE: The details of the microscope set-up have been previously described6,7.
<ol>
	<li>Microscope
	<ol>
		<li>Turn on the heating blanket. Turn on the argon laser for 488 nm excitation and the solid-state 405 nm, 561 nm, and 640 nm lasers. Turn on the microscope, the spinning disk control unit, the AOTF, the laser control unit, and the camera controller.</li>
		<li>Open the microscope shutter, which turns the LED red. Turn on the computer and camera software.</li>
	</ol>
	</li>
	<li>Imaging Platform
	<ol>
		<li>Ensure that the stage insert is clean. Otherwise, clean with soap and water and then dry.</li>
		<li>Take two coverslips and use lab tape to secure them on the insert. Place the insert on the stage and clean it with alcohol wipes.</li>
		<li>Using lab tape, attach the heating blanket to the right side of the stage.</li>
		<li>Verify the integrity of the rubber diaphragm on the nose cone and change it if necessary. Position and fix the nose cone for isoflurane anesthesia delivery to the animal by using gauze and lab tape.</li>
	</ol>
	</li>
	<li>Isoflurane Anesthesia System
	<ol>
		<li>Refill the isoflurane tank.</li>
		<li>Turn on the vacuum and adjust to 1.2 L/min.</li>
		<li>Add distilled water to the nebulizer and connect the nebulizer to the isoflurane system.</li>
		<li>Turn on the oxygen analyzer and connect it to the isoflurane system. Turn on the oxygen tank and ensure that the oxygen analyzer shows 100%. Adjust the O2 flow to 0.2 L/min.</li>
		<li>Turn on the nitrogen tank and adjust the flow at 0.8 L/min. Ensure that the oxygen analyzer shows 21%.</li>
	</ol>
	</li>
	<li>Preparation of Surgery Tools and Platform
	<ol>
		<li>Clean 2 pairs of dissection forceps and scissors with soap and water.</li>
		<li>Turn on the hot bead sterilizer and let it reach 250 &#176;C. Sterilize the surgery tools for 30 sec. Let them cool off.</li>
		<li>Place a lab soaker on the surgery bench.</li>
		<li>Use a Styrofoam lid as a surgical platform. Cover with a piece of lab soaker. Prepare a second piece of lab soaker to change after shaving the mouse.</li>
		<li>Place the nose cone with the line of anesthesia on the covered Styrofoam lid, and attach it to the Styrofoam lid (not on the lab soaker) with some tape and use two needles on both sides of the nose cone to keep it in place.</li>
		<li>Assemble betadine, alcohol wipes, sterile gauze, super glue, microscope slides, electric shaver, and 4 pieces of lab tape.</li>
	</ol>
	</li>
</ol>
3. Preparation of Injections
<ol>
	<li>Under the hood, prepare an insulin syringe (28 G x &#189; in) with 7 &#181;l of stock AF647-conjugated Ly-6G (Gr1) antibody (1 mg/ml) into 100 &#181;l of 2,000 kDa rhodamine-dextran (4 mg/ml) for retro-orbital injection. Retro-orbital injection is recommended in ApcMin/+ mice since anemia that develops in these animals makes the tail veins difficult to detect through the skin.</li>
	<li>Prepare a second insulin syringe with 1 mg/kg atropine diluted into 150 &#181;l PBS for subcutaneous (s.c.) injection before imaging to dampen peristaltic movements of the intestine.</li>
</ol>
4. Preparation of the Intestine for Imaging
<ol>
	<li>Verify that the anesthesia line to the induction chamber is open, and the lines to the surgical table and microscope are closed.</li>
	<li>Transfer the animal to the anesthesia chamber. Close the anesthesia chamber and turn on the isoflurane to 4% (with 21% O2).</li>
	<li>When the mouse breathes deeply and slowly (after 5-6 min), transfer it to the surgical stage on its flank and inject the Gr1 antibody solution and/or dextran solution retro-orbitally.</li>
	<li>Open the anesthesia line linked to the surgical platform. Close the line to the anesthesia chamber and put the mouse on its back with its nose in the anesthesia cone.</li>
	<li>Reduce the isoflurane concentration from 4% to 2.5%.</li>
	<li>Verify that the mouse is well anesthetized by pinching its footpad and testing the corneal reflex.</li>
	<li>Add ophthalmic lubricating&#160;ointment on eyes to prevent dryness while under anesthesia.</li>
	<li>Secure the limbs of the mouse to the surgical stage by adding lab tape.</li>
	<li>Remove the hair from the ventral surface by using an electric shaver. Also remove the hair from the left hind limb to position the oximeter before acquisition. Remove the lab soaker from the surgical stage and replace with a clean one to avoid hair contamination.</li>
	<li>Disinfect the ventral surface with alcohol wipes and betadine.</li>
	<li>Inject the atropine solution (from 3.2) s.c. in one flank of the mouse.</li>
	<li>Make a 1 cm ventral midline incision through the skin and the peritoneum by using scissors and one pair of forceps. Carefully pull out 3-4 cm of intestine and identify one or several tumors to image.</li>
	<li>Take a glass microscope slide and position a loop of the intestine on it with a tumor at the top.</li>
	<li>Add some super glue to a few locations along the intestine to attach it to the slide. Wait one min for the glue to dry. Use a marker to draw a line on the slide pointing to the tumor in order to facilitate its localization with the confocal.</li>
</ol>
5. Positioning the Mouse on the Stage and Preparation for Image Acquisition
<ol>
	<li>Remove lab tape from the limbs.</li>
	<li>Open the anesthesia line to the microscope stage and close the one to the surgical platform.</li>
	<li>Transfer the mouse carefully to the microscope stage with its ventral side down and its nose in the nose cone. Secure the anesthesia line with some lab tape.</li>
	<li>Re-position the mouse and/or the slide in order to have the intestine in the center of one of the cover-slipped windows. Gently tape down the slide with lab tape.</li>
	<li>Attach the oximeter probe to the left limb of the mouse to follow its heart and respiratory rate and arterial oxygen saturation levels.</li>
	<li>Cover the mouse with the heating blanket to avoid hypothermia.</li>
	<li>Decrease the isoflurane concentration from 2.5% to 1-1.5% for imaging.</li>
</ol>
6. Acquisition of Images by Using &#181;Manager Software
<ol>
	<li>Increase CSUX speed to 2,500 rpm to improve the image quality in the Configuration Settings.</li>
	<li>Use the Objective dropdown menu to choose 10X 0.5 NA or 20X 0.75 NA Fluar lens objective.</li>
	<li>Choose the laser 405 nm from Color dropdown menu.</li>
	<li>Click on Live and adjust the focus with the microscope.</li>
	<li>Click on Auto to automatically adjust maximum and minimum pixel intensities of the display image based on the extreme pixel values in the image. A histogram of display image&#39;s pixel intensities is presented in the graph in the lower part of the Main Window.</li>
	<li>Adjust the camera exposure in the Piper Control Panel window by changing the number of clocks (One clock is 33.333 msec).</li>
	<li>If the signal is too bright with the lowest exposure, decrease laser intensity using the microscope control unit.</li>
	<li>Check the signal intensity for 488 nm, 561 nm and 640 nm laser lines. Adjust laser intensity with the laser&#8217;s own control unit (488 nm and 561 nm) or the microscope control unit.</li>
	<li>In the Multi D Acquisition window, check the Time points box and type in number of time points and desired time interval.</li>
	<li>Check the Z-stacks box, choose &#8220;relative Z&#8221; and define Z-start and Z-end relative to the current position. 
	NOTE: For 10X or 20X objective, start with 3 Z planes, 8 &#181;m or 4 &#181;m apart respectively.</li>
	<li>Check the Multiple positions (XY) box for imaging several locations, then click Edit position list, mark 4 to 6 different positions.</li>
	<li>Check the Channels box and add channels from New and select lasers lines from the dropdown menu and type in exposure for each channel (in multiples of 33.333 msec).</li>
	<li>Check Save images in the &#8220;Multi D acquisition&#8221; window.</li>
	<li>Select a folder and all the images acquired will be automatically saved in this folder with the name prefix given. 
	NOTE: Each continuous acquisition will be saved in a separate subfolder as individual tiff files for each Z slice in each color at each time point.</li>
	<li>Save all the settings by clicking Save settings in the Multi D Acquisition window.</li>
	<li>Click Acquire in the Multi-dimensional Acquisition window to start acquisition.</li>
	<li>If the oximeter probe is not used or stops working well (when pulse is not stable or hard to detect), check the efficiency of anesthesia every 15 min during the imaging procedure by pinching the footpad and checking the corneal reflex. Adjust the anesthesia if the mouse is responsive to these stimuli.</li>
	<li>After acquisition, euthanize the mouse by increasing the isoflurane concentration to 5%. When no breathing movements are detectable, remove the mouse from the stage and perform a cervical dislocation.</li>
</ol>
7. Analysis of Images by Using Imaris Software
<ol>
	<li>Conversion to .ims Files (Supplementary Figure 1A)
	<ol>
		<li>Open the camera software, click on File then Batch Converter.</li>
		<li>Click on Add files and choose the first file of the first position. Repeat for each position.</li>
		<li>For each file uploaded, click on Settings and verify that the settings &#8220;t&#8221; for Time, &#8220;c&#8221; for Channels and &#8220;z&#8221; for Z-stacks appear in that order.</li>
		<li>Save in the desired folder. Browse and create a new file as converted.</li>
		<li>Click on Set for all and Start.</li>
	</ol>
	</li>
	<li>Adjustments (Supplementary Figure 1B)
	<ol>
		<li>Go into the specific Converted folder and open the first file.</li>
		<li>In Display Adjustment window, adjust the background signal cutoff by modifying the Min number for each channel&#160;if necessary.</li>
		<li>If necessary, adjust the signal intensity by modifying the Max number (the lower the Max number, the higher the intensity of the signal). 
		NOTE: Adjustments of the signal intensity/contrast enable the highlight of a cell behavior or a compartment in order to have a nicer visualization. It does not change the result itself.</li>
		<li>Click on Edit and Image Properties.</li>
		<li>Change the x, y, and z values (for a 10X objective, x and y = 0.712, z = 8 &#181;m; for a 20X objective&#160;: x and y = 0.36, z = 4 &#181;m).</li>
		<li>Click on All Equidistant to adjust time points. Add the start date and start time of the acquisition. Add the end date and end time of the acquisition (Supplementary Figure 1C).</li>
		<li>Click on Play button to watch the movie.</li>
		<li>Click Edit and Delete time points to delete blurry images.</li>
		<li>To join two separate acquisitions together into one movie, go to the last time point of the first movie and click on Edit&#160;&#62;&#160;Add time points to add the second file.</li>
	</ol>
	</li>
	<li>Saving as a movie (Supplementary Figure 1D)
	<ol>
		<li>Click on Record, choose the .mov extension and a compression factor of 0.</li>
		<li>Click on Save.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

In this paper, a detailed protocol is described for spinning disk confocal imaging of myeloid cell dynamics in intestinal tumors for several hours in a live animal, imaged from the serosal side of the gut.
To avoid inflammation and to have optimal physiologic conditions, imaging of the intestine has to be done on the intact organ. However, imaging from the serosal side of the intestine is challenging, since the light has to go through different tissue layers such as smooth muscle before reachi...

Overwhelming evidence now demonstrates that the tumor microenvironment, consisting of heterogeneous cell populations, including fibroblasts, endothelial cells, immune and inflammatory cells, extracellular matrix, and soluble factors, plays a crucial role in the initiation and progression of solid tumors by contributing to almost all hallmarks of cancer1. Indeed, during tumor progression, there are constant dynamic interactions between transformed cancer cells and stromal cells that evolve to generate a microenvironment favorable to malignancy2. Among the immune cells that infiltrate the tumor microenvironment, myeloid cells are the most abundant3. Consisting of tumor-associated macrophages (TAM), myeloid-derived suppressor cells (MDSCs), dendritic cells (DC) and neutrophils (PMNs), myeloid cells are recruited from bone marrow and progressively infiltrate tumors, releasing cytokines, growth factors and proteases which can promote tumor growth and spread4. The crosstalk between cancer cells and myeloid cells is complex but dynamic. Thus the understanding of the nature of their interactions is crucial for determining why these cells promote cancer progression instead of participating in an anti-tumor immune response, and may help to find new targets to control it.
Direct observation by intravital microscopy provides information on cell dynamics within the tissues of live mice5. A four-color, multi-region, micro-lensed spinning disk confocal system was designed to study stromal cells within mammary tumors6. This approach enables long-term continuous imaging and includes several advantages such as (a) rapid images acquisition to minimize motion artifacts, (b) long-term anesthesia, (c) four color acquisition to follow different cell types, (d) fluorescent labeling of different tumoral components, and (e) observation of different tumor microenvironments within the same mouse to avoid mouse to mouse variability7-9. With this technology, different cell behaviors have been reported in the mammary tumor virus (MMTV) promoter-driven polyoma middle T oncogene (PyMT) model that displays progressive stages of tumorigenesis. Regulatory T-lymphocytes (Tregs, visualized by the Foxp3EGFP transgene) migrate preferentially in proximity to blood vessels whereas DCs (CD11c-DTR-EGFP), carcinoma-associated fibroblasts (Fsp1+/+-EGFP) and myeloid cells (c-fms-EGFP) exhibit higher motility at the tumor periphery than within the tumor mass. In the condition of acute systemic hypoxia, cells migrated differently: Tregs stop migrating in contrast to myeloid cells that continue to move6. In addition, in the same mouse model, it has been shown that doxorubicin sensitivity changes with tumor stage, drug distribution is related to drug response, and doxorubicin treatment leads to CCR2-dependent recruitment of myeloid cells to tumors. Thus, live imaging can also be used to gain insights into drug responses in&#160;situ and the biology of chemoresistance10,11.
Adenomatous polyposis coli (Apc) gene mutations commonly occur in human colorectal adenomas and carcinomas12 and mutation of a single copy of the Apc gene results in familial adenomatous polyposis (FAP), which confers an extremely high risk for colon cancer13. The mouse strain ApcMin/+ carries a truncation mutation at codon 850 of the Apc gene and spontaneously develops multiple intestinal adenomas all over the small intestine14-16. Long-term intravital imaging of the intestine is challenging because of the invasiveness of the procedure, since opening the peritoneal cavity is necessary for access to the intestine. Short-term live imaging studies have been previously published on healthy intestine17,18, but long-term direct observation of intestinal tumors has not been reported. A surgical procedure has been designed and refined to visualize tumors through the serosal surface of the intestine, using the intravital spinning disk microscopy system previously used to image breast tumors6,10. In this paper, a protocol is described that allows one to follow the behavior of myeloid cells within the tumors in the small intestine by using ApcMin/+ mice.

<table><tbody><tr><td>&lt;em&gt;Apc&lt;sup&gt;Min/+&lt;/sup&gt;&lt;/em&gt; mice</td><td>Jackson Laboratory</td><td>2020</td><td></td></tr><tr><td>ACTB-ECFP mice</td><td>Jackson Laboratory</td><td>3773</td><td></td></tr><tr><td>cfms-EGFP mice</td><td>Jackson Laboratory</td><td>18549</td><td></td></tr><tr><td>2,000 kDa Dextran, rhodamine-conjugated</td><td>Invitrogen</td><td>D7139</td><td></td></tr><tr><td>Isoflurane</td><td>Butler Animal Health Supply</td><td>29450</td><td></td></tr><tr><td>Nitrogen</td><td>UCSF</td><td></td><td></td></tr><tr><td>Oxygen</td><td>UCSF</td><td></td><td></td></tr><tr><td>1x PBS</td><td>UCSF cell culture facility</td><td></td><td></td></tr><tr><td>Saline Buffer</td><td>UCSF cell culture facility</td><td></td><td></td></tr><tr><td>Anti-mouse Ly-6G (GR1) antibody AF647</td><td>UCSF Monoclonal antibody core</td><td></td><td>Stock 1 mg/ml. Use at 7 &amp;mu;g/mouse</td></tr><tr><td>Atropine</td><td>LARC UCSF</td><td></td><td>Use at 1 mg/kg mouse</td></tr><tr><td>Alcohol wipes</td><td>Becton Dickinson</td><td>326895</td><td></td></tr><tr><td>28 G x 1/2 in. insulin syringe</td><td>Becton Dickinson</td><td>329465</td><td></td></tr><tr><td>Remium cover glass</td><td>Fisher Scientific</td><td>12-548-5M</td><td>24x50-1</td></tr><tr><td>Betadine</td><td>LARC UCSF</td><td></td><td></td></tr><tr><td>Heat blanket</td><td>Gaymar Industries</td><td></td><td></td></tr><tr><td>Hot bead sterilizer</td><td>Fine Science Tools</td><td>18000-45</td><td>Turn ON 30 min before use</td></tr><tr><td>Cotton tipped apllicators 6-inch</td><td>Electron Microscopy Sciences</td><td>72310-10</td><td></td></tr><tr><td>Anesthesia system</td><td>Summit Anesthesia Support</td><td></td><td></td></tr><tr><td>Inverted microscope</td><td>Carl Zeiss Inc</td><td>Zeiss Axiovert 200M</td><td></td></tr><tr><td>Stage insert</td><td>Applied Sientific Instrumentation</td><td></td><td></td></tr><tr><td>Mouse Ox oximeter, software and sensors</td><td>Starr Life Sciences</td><td>MouseOx</td><td></td></tr><tr><td>Nebulizer</td><td>Summit Anesthesia Support</td><td></td><td></td></tr><tr><td>Imaris</td><td>Bitplane</td><td></td><td></td></tr><tr><td>&amp;mu;Manager</td><td>Vale lab, UCSF</td><td></td><td>Open-source software</td></tr><tr><td>ICCD camera</td><td>Stanford Photonics</td><td>XR-Mega-10EX S-30</td><td></td></tr><tr><td>Spinning disk confocal sacan-head</td><td>Yokogawa Corporation</td><td>CSU-10b</td><td></td></tr></tbody></table>

real-time imaging of myeloid cells dynamics in apcmin/+ intestinal tumors by spinning disk confocal microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

By using spinning disk confocal microscopy, non-tumoral and tumoral tissues in the small intestine of ApcMin/+;ACTB-ECFP;c-fms-ECFP mice can be visualized from the serosal surface. After imaging, camera software is used to analyze and adjust the acquisition (Supplementary Figure 1). After intravenous (i.v.) injection of fluorescent 2,000 kDa dextran-rhodamine and a Ly-6G 647 conjugated antibody, blood vessels and PMNs can be detected respect...

Watch this Scientific Journal Video about Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy at JoVE.com

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

real-time-imaging-myeloid-cells-dynamics-apcmin-intestinal-tumors

Research

JoVE Journal

Immunology and Infection

9.7K Views.  INSERM U661, Functional Genomic Institute. By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model. 

Video: Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Murine Pancreatic Imaging Using Implanted Stabilized Window

Stabilized Window for Intravital Imaging of the Murine Pancreas

The physiology and pathophysiology of the pancreas are complex. Diseases of the pancreas, such as pancreatitis and pancreatic adenocarcinoma (PDAC) have high morbidity and mortality. Intravital imaging (IVI) is a powerful technique enabling the high-resolution imaging of tissues in both healthy and diseased states, allowing for real-time observation of cell dynamics. IVI of the murine pancreas presents significant challenges due to the deep visceral and compliant nature of the organ, which make it highly prone to damage and motion artifacts. 
Described here is the process of implantation of the Stabilized Window for Intravital imaging of the murine Pancreas (SWIP). The SWIP allows IVI of the murine pancreas in normal healthy states, during the transformation from the healthy pancreas to acute pancreatitis induced by cerulein, and in malignant states such as pancreatic tumors. In conjunction with genetically labeled cells or the administration of fluorescent dyes, the SWIP enables the measurement of single-cell and subcellular dynamics (including single-cell and collective migration) as well as serial imaging of the same region of interest over multiple days. 
The ability to capture tumor cell migration is of particular importance as the primary cause of cancer-related mortality in PDAC is the overwhelming metastatic burden. Understanding the physiological dynamics of metastasis in PDAC is a critical unmet need and crucial for improving patient prognosis. Overall, the SWIP provides improved imaging stability and expands the application of IVI in the healthy pancreas and malignant pancreas diseases.

Author Spotlight: Revolutionizing Pancreatic Disease Understanding Through Advanced Intravital Imaging 

We present a protocol for the surgical implantation of a stabilized indwelling optical window for subcellular-resolution imaging of the murine pancreas, allowing serial and longitudinal studies of the healthy and diseased pancreas.

The physiology and pathophysiology of the pancreas are complex. Diseases of the pancreas, such as pancreatitis and pancreatic adenocarcinoma (PDAC) have ...

Cancer Research

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 ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

Bioengineering

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 ...

Medicine

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.

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic ...

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal ...

Measurement of Microtubule Dynamics by Spinning Disk Microscopy in Monopolar Mitotic Spindles

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a genetically encoded marker for the positive ends of microtubules (EB3 labelled with tdTomato fluorescent protein) and high-speed, high-resolution, live-cell imaging using spinning disk confocal microscopy. Cell cycle synchronization and increased density of microtubules are achieved by inhibiting centrosomal separation in mitotic cells, and analysis of growth is performed using open-source U-Track software. The use of a bright and red-shifted fluorescent protein, in combination with the lower laser power and reduced exposure time required for spinning disk microscopy reduce phototoxicity and the probability of light-induced artifacts. This allows for imaging a larger number of cells in the same preparation while maintaining the cells in a growth medium under standard culture conditions. Because the analysis is performed in a supervised automatic fashion, the results are statistically robust and reproducible.

Here we present a robust and detailed method of microtubule dynamics analysis in cells synchronized in prometaphase using live-cell spinning disk confocal microscopy and MATLAB-based image processing.

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a ...

Biology

Real-Time Analysis of Resident T-cell Migration in Different Tumor Regions Using Confocal Microscopy

Source: Peranzoni, Elisa, et al. <a href="https://app.jove.com/v/55709">Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy.</a> J. Vis. Exp. (2017).
This video describes the imaging of resident tumor-infiltrating CD8 T cells labeled with fluorescently coupled antibodies within human lung tumor slices. This technique permits real-time analyses of CD8 T cell migration using confocal microscopy.

Real-time Imaging of Myeloid Cells Dynamics in Apc^Min/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

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