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 reaching the epithelium. Focusing on the epithelium can be difficult in some areas, particularly in the actin-ECFP reporter mice since actin is highly expressed by smooth muscle cells. Although two-photon microscopy has the benefit of deeper tissue penetration as compared to confocal microscopes, we found that the spinning disk confocal microscope is powerful enough to obtain a good visualization of the intestinal epithelial structure. The advantages of spinning disk microscopy include the capacity for very rapid acquisition and unique sensitivity without sacrificing confocal resolution. These qualities allow the observation of dynamic processes even if some time points have to be deleted due to movement artifacts, minimize photodamage, and enable the imaging of relatively weakly fluorescent molecules.
Imaging intestine from the serosal side can also be challenging because of the endogenous peristaltic movements of this organ. In this protocol, a s.c. injection of atropine is given before imaging, and it efficiently decreases the movement. However, it is important to note that peristaltic movement is more extensive in the large intestine, but is not well inhibited by atropine treatment, rendering imaging quite difficult in this part of the gut. The smooth muscle layer in the colon is somewhat more prominent compared to the small intestine and appears to be more contractile. Therefore, it is almost impossible to focus on the epithelium in the actin-ECFP reporter mice. Moreover, in addition to these endogenous movements, organ drifting can occur, but in this case posterior drift correction can be performed by using the camera software.
Another major challenge in invasive imaging experiments is to keep the mouse alive and healthy as long as possible under anesthesia. The intestine is reached by opening the peritoneal cavity. Thus the procedure is more invasive than that used for mammary tumor imaging, which can be done for up to 40 hr because of the integrity of the peritoneum. The maximum acquisition done with the peritoneal cavity opened was 6 hr, but the mouse was still alive with no obvious signs of distress, so even longer acquisition may be feasible.
In this study myeloid cell behavior can be followed and analyzed within the intestinal tumor by using fluorescent reporter mice and also fluorochrome-conjugated tracers or antibodies. This approach can also be used for observing other cell types such as Tregs, dendritic cells or fibroblasts by using Foxp3-EGFP or CD11c-DTR-EGFP, or Fsp1-EGFP reporter mice respectively, as it has been used in the mammary tumor study6. Also, fluorescent antibodies against other cell populations than neutrophils can be injected i.v. In addition, cell behavior can also be analyzed in different conditions, such as hypoxia. Such experiments may give new insights into the behavior of those stroma cells after treatment with chemotherapy or targeted therapy. Finally, this protocol can be used in other mouse models of intestinal cancer and in different stages of tumor progression. ApcMin/+ mice develop intestinal adenomatous polyps only, which do not progress to malignancy because the mice have a shortened lifespan. It will be interesting to study the dynamic of myeloid cells in other intestinal cancer models with more aggressive and invasive tumors such as mice carrying mutations in K-ras19-21or Smad422 genes in addition to the Apc mutation.

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 respectively (Figure 1). Peyer&#8217;s patches that are aggregations of lymphoid tissue and full of myeloid cells can be seen easily (Figure 1A). The crypts of the small intestine are surrounded by blood vessels and myeloid cells (Figure 1A and 1B). Tumoral tissue is often more vascularized and more infiltrated by myeloid cells, and the structure of the crypts is less well organized (Figure 1A and 1B) depending on the size of the tumor. Acquisition can be done by using a 10X 0.5 NA (Figure 1A) and 20X 0.75 NA Fluar lenses (Figure 1B) but we obtained the best performance with the 10X 0.5 NA Fluar lens which was bright, allow the acquisition of a large region (676 x 676 &#181;m) and could provide images from up to 70 &#181;m into the tissue. The smooth muscle layer expressing actin-ECFP can sometimes render the focus on the intestinal epithelium difficult (Figure 1B, right panel). Myeloid cells dynamics in tumors can be followed in real time for a few hours as shown in the Animated/Video Figure 2. Whereas neutrophils (Gr1+ cells) circulate in blood vessels and patrol the tissues, other myeloid cells, mostly macrophages, surround the crypts and are less motile (Animated/Video Figure 2).
<img alt="Figure 1" src="/files/ftp_upload/51916/51916fig1highres.jpg" /> 
Figure 1. Representative pictures obtained with the spinning disk confocal of non-tumoral and tumoral tissue from ApcMin/+ mice.&#160; (A) Four-color pictures (10X Fluar, 0.5 NA lens) of the small intestine epithelium from the serosal surface of ApcMin/+;ACTB-ECFP;cfms-EGFP mouse that received i.v. fluorescent 2,000 kDa dextran-rhodamine (red) and a Ly-6G 647 conjugated antibody (white). The left panel shows the non-tumoral epithelium and the right panel shows a tumor from the same ApcMin/+;ACTB-ECFP;cfms-EGFP mouse. Myeloid cells are green and epithelial cells are blue. A cluster of c-fms+ cells is seen in the non-tumoral intestinal tissue in a Peyer&#8217;s patch (white arrow), scale bar = 100 &#181;m. (B) Four-color pictures (20X Fluar, 0.5 NA lens) of the small intestine epithelium from the serosal surface of a ApcMin/+;ACTB-ECFP;cfms-EGFP tumor. In the left panel, several double-positive Gr1+ c-fms+ cells are detected in the tumoral tissue. The right panel shows a picture of the smooth muscle layer expressing actin-ECFP that can render the focus on the intestinal epithelium difficult, scale bar = 50 &#181;m.
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51916/Animated-Video_Figure_2.mov" target="_blank">Animated/Video Figure 2</a>. Myeloid cell dynamics in a 3 mm adenoma from an ApcMin/+ mouse. Four color time-lapse of the small intestine epithelium (10X Fluar, 0.5 NA lens) from the serosal surface of a ApcMin/+;ACTB-ECFP;cfms-EGFP mouse co-injected with fluorescent 2,000 kDa dextran-rhodamine (red) and a Ly-6G 647 conjugated antibody (white). Myeloid cells are green and epithelial cells are blue. Myeloid cells around the crypts are not moving. Some neutrophils (Gr1+ cells in white) are patrolling in the tissues. This is a 2 hr acquisition and the movie has been sped up 295 times.
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51916/Supplementary Figure 1.tif" target="_blank">Supplementary Figure 1</a>. Screen shots of camera software for acquisition analysis. (A) First, the files need to be converted into .ims extension. (B) Then, display images can be adjusted, such as specific signals can be increased and background decreased. (C) Date and Time need to be adjusted in order to get a real-time movie. (D) The acquisition can be saved as a movie.

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

9.6K 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

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the tumor microenvironment and respond adequately to tumor antigens. The recent development of improved imaging approaches, such as 2-photon microscopy, and the use of powerful mouse tumor models have shed light on some of the mechanisms that regulate anti-tumor T cell activities. Whereas such systems have provided valuable insights, they do not always predict human responses. In human, our knowledge in the field mainly comes from a description of fixed tumor samples from human patients, as well as in vitro studies. However, in vitro models lack the complex three-dimensional tumor milieu and, therefore, are incomplete approximations of in vivo T cell activities. Fresh slices made from explanted tissue represent a complex multi-cellular tumor environment that can act as an important link between co-cultured studies and animal models. Originally set up in murine lymph nodes1 and previously described in a JoVE article2, this approach has now been transposed to human tumors to examine the dynamics of both plated3&#160;as well as resident&#160;T cells4.
Here, a protocol for the preparation of human lung tumor slices, immunostaining of resident CD8 T and tumor cells, and tracking of CD8 T lymphocytes within the tumor microenvironment using confocal microscopy is described. This system is uniquely placed to screen for novel immunotherapy agents favoring T cell migration in tumors.

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

This protocol describes a method to image 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.

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the ...

Cancer Research

Tracking Mouse Bone Marrow Monocytes In Vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

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 ...

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

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fc&#947; receptor (Fc&#947;R)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to Fc&#947;Rs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fc&#947; receptor- or complement receptor-mediated phagocytosis.

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...

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.remotexs.ntu.edu.sg/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

Summary

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Chapters in this video

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

Tracking Mouse Bone Marrow Monocytes In Vivo

Stabilized Window for Intravital Imaging of the Murine Pancreas

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

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

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

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

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

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