This research was supported by the Department of Defense Breast Cancer Research Program under award number (A.S.H, W81XWH-13-1-0010), NIH CA100324, &#160;PPG CA100324,&#160;and the Integrated Imaging Program.

All procedures described must be performed in accordance with guidelines and regulations for the use of vertebrate animals, including prior approval by the Albert Einstein College of Medicine Institutional Animal Care and Use Committee.
1. Generating Fluorescently Labeled Tumors and Tumor-associated Macrophages
<ol>
	<li>Generate fluorescently labeled tumor cells by crossing the spontaneous, autochthonous, genetically engineered mouse mammary cancer model where the mouse mammary tumor virus long terminal repeat drives the polyoma middle T antigen (MMTV-PyMT) with transgenic mice with fluorescent reporters [i.e., enhanced green fluorescent protein (EGFP), enhanced cyan fluorescent protein (ECFP) or Dendra2]8,9.</li>
	<li>Fluorescently label macrophages in PyMT animals with fluorescently labeled tumor cells by crossing genetically engineered mouse models with myeloid- and macrophage- specific fluorescent reporters [i.e., MacGreen15 or MacBlue mice Csf1r-GAL4VP16/UAS-ECFP16] .</li>
	<li>Alternatively, generate fluorescently labeled tumors through orthotopic transplantation of fluorescently labeled PyMT tumor cells (syngeneic), human breast cancer cell lines or primary human patient derived tumors (xenografts)11,17.
	<ol>
		<li>Implant fluorescently labeled PyMT tumor cells into syngeneic mice with fluorescent protein-labeled myeloid cells to generate animals with fluorescently labeled tumor cells and myeloid cells13.</li>
	</ol>
	</li>
	<li>Inject 100 &#181;l of 10 mg/kg fluorescent 70 kDa dextran by intravenous (i.v.) tail vein into severe combined immunodeficiency (SCID) mice with xenograft tumors of human cell lines or patient-derived primary tumors to fluorescently label macrophages 2 hr prior to the start of intravital imaging.</li>
</ol>
2. Microscope Setup and Imaging Preparation
Note: This procedure describes the set-up for intravital imaging on a multiphoton microscope8.
<ol>
	<li>Turn on all microscope and laser components including two-photon lasers and the detectors.</li>
	<li>Turn on the heating box to 30 &#176;C to pre-warm the stage. This step is critical for maintaining the physiological temperature of the animal.</li>
	<li>For use on an inverted microscope place the custom-made stage insert on the microscope stage. The custom insert is a sheet of 1/8&#34; thick aluminum machined to fit in the stage insert space and with a 1&#34; diameter through-hole in the center for imaging.
	<ol>
		<li>Wipe the microscope stage and stage insert with 70% ethanol and air dry.</li>
		<li>Place a large drop of water on the 20X, 1.05&#160;NA microscope objective to maintain optical contact with the cover glass.</li>
		<li>Place cover glass (#1.5 thickness) over the imaging port on the microscope stage insert. Secure in place with lab tape.</li>
		<li>Use a hole punch to punch a 2 cm x 2 cm hole in a flexible rubber pad and place the rubber pad on the microscope stage with the hole in the pad aligned with the hole in the custom stage insert.</li>
	</ol>
	</li>
</ol>
3. Preparation of Tail Vein Catheter and Reagents for Injection during Imaging
<ol>
	<li>Cut a 30 cm length of polyethylene (PE) tubing.</li>
	<li>Using forceps, move the metal needle of a 31 G needle back and forth until it breaks off of the plastic fitting.</li>
	<li>Using forceps, insert the blunt end of the detached needle into the PE tubing.</li>
	<li>Insert a 31 G needle into the other end of the PE tubing.</li>
	<li>Fill a 1 cc syringe with sterile phosphate buffered saline (PBS) and insert it into the 31 G needle and flush all air out of the tubing and needle with PBS. PBS is used for maintaining hydration and blood osmolarity9,18, however isotonic saline may also be used.</li>
	<li>Fill a 1 cc syringe with 200 &#181;l of 3 mg/kg 155 kDa dextran-tetramethylrhodamine (TMR) or quantum dots for vascular labeling.</li>
	<li>Prepare any other injectable proteins such as a 165 amino acid isoform of vascular endothelial growth factor A (VEGFA165) in a 1 cc syringe and place on ice. Inject 0.2 mg/kg VEGFA16519 at a concentration of 0.05 mg/ml.</li>
</ol>
4. Insertion of the Indwelling Tail Vein Catheter
<ol>
	<li>Place the animal under anesthesia using an induction chamber with 5% isoflurane with O2 as the carrier gas.</li>
	<li>Transfer the mouse to the surgical platform once it is fully anesthetized and does not respond to a toe pinch.</li>
	<li>Open isoflurane anesthesia line to the surgical platform, close the anesthesia line to the induction chamber and place the nose cone over the animal&#39;s snout. Reduce the isoflurane from 5% to 2.5%.</li>
	<li>Apply ophthalmic ointment to the eyes of the mouse to prevent dryness during anesthesia.</li>
	<li>Heat the animal under the heating lamp for an additional 2 min to increase circulation in the tail as circulation slows with deep anesthesia.</li>
	<li>Sterilize the mouse tail vein with 70% ethanol.</li>
	<li>Insert the tip of the 31 G needle from the catheter constructed in Section 3 into the lateral tail vein of the animal and push the needle in 2-3 mm.</li>
	<li>Pull back on the syringe to see blood, ensuring the catheter is placed in the tail vein.</li>
	<li>Use a 1 cm length of lab tape placed over the needle to hold the needle in place parallel to the vein along the length of the tail.</li>
</ol>
5. Skin Flap Surgical Procedure to Expose the Mammary Tumor
<ol>
	<li>With the animal on the surgical platform swab the tumor and the ventral surface with 70% ethanol.&#160;Note: As described, this procedure is not performed completely aseptically.&#160; For long imaging sessions where infection can become a confounding factor, it is recommended to use proper aseptic technique.</li>
	<li>Using sterile forceps, lift the ventral skin and with sterile scissors make a subcutaneous incision along the ventral midline approximately 1 cm in length. Avoid puncturing the peritoneum during the incision.</li>
	<li>Gently cut the connective tissue attaching the mammary gland and tumor away from the peritoneum to expose the mammary tumor.</li>
	<li>Using scissors and forceps, gently remove and cut away the fascia and fat from the exposed surface of the tumor while maintaining the integrity of the tumor vasculature and minimizing bleeding. This step is critical in maintaining the tumor architecture and vasculature supply of the tumor.</li>
</ol>
6. Animal Preparation for Microscopy
<ol>
	<li>Transfer the animal to the pre-warmed imaging stage with the exposed tumor placed on the coverslip on the stage insert over the microscope objective for imaging. Take care to transfer the tail vein catheter gently with the animal so as not to dislodge the needle.</li>
	<li>Place the anesthesia nose cone over the animal&#39;s snout to maintain anesthesia set to 3% isoflurane.</li>
	<li>Position the animal so that the tumor rests in the hole of the rubber pad and makes contact with the glass coverslip.</li>
	<li>Use two rubber pads to gently hold the tumor in place and fix them to the microscope stage with lab tape to reduce movement during image acquisition.</li>
	<li>Fill the chamber from the rubber pad with PBS to keep the tissue hydrated and maintain optical contact with the coverslip.</li>
	<li>Start monitoring the animal&#39;s vital signs with a pulse oximeter probe. Attach a clip sensor to the thigh of the animal. Alternatively a collar that is configured to fit around the neck can be used.</li>
	<li>Place the heating box over the animal to maintain 30 &#176;C 20.</li>
	<li>Slowly reduce the level of isoflurane to 0.5-1% to maintain anesthesia and maintain blood flow.</li>
</ol>
7. Image Acquisition and Injection of Fluorescent Dyes and Injectable Proteins
<ol>
	<li>Using the microscope eyepiece focus on the fluorescent tumor cells on the surface of the tumor.</li>
	<li>Select an area of interest by finding areas with flowing blood vessels. The selection of an area of interest with flowing blood vessels is critical for assessing tumor vasculature.</li>
	<li>Once a region of interest has been selected switch the microscope into multiphoton imaging mode.</li>
	<li>Set the upper and lower limits of a z-series. Set the upper limit of the z-series by using the focus adjuster to move the objective to the desired start location and clicking on the Z position &#34;Top&#34; button. Determine the upper limit of the z-series by visualizing the collagen fiber network at the surface of the tumor in the second harmonic generation (SHG) channel and observing when no cells but only collagen fibers are visible.
	<ol>
		<li>Set the lower limit of the z-series by moving the objective to the desired imaging depth (typically 50 - 150 &#181;m) and clicking the Z position &#34;Bottom&#34; button.</li>
		<li>Set the step size by typing the desired value into the step size field. Determine step size based on considerations for resolution and acquisition time (typically 2 &#181;m is used for high-resolution 3D reconstruction and 5 &#181;m otherwise).</li>
	</ol>
	</li>
	<li>Set the time-lapse interval by switching to the Time-Lapse panel and entering the desired lapse time into the Time-Lapse field. For optimal temporal resolution set the time interval to 0 sec for continuous imaging. Note: For long time-lapse imaging it is recommended to set the time interval to 10 sec between acquisitions to replenish the objective immersion liquid.</li>
	<li>Once the parameters for imaging have been determined, slowly inject 155 kDa dextran-TMR.
	<ol>
		<li>Remove the syringe with PBS in the tail vein catheter and replace with the syringe containing 155 kDa dextran-TMR taking care not to introduce any bubbles into the line.</li>
		<li>Slowly inject 155 kDa dextran-TMR into the mouse, with a maximum volume of 200 &#181;l, and replace the syringe with syringe containing PBS. CRITICAL STEP: Perform the injection slowly and take precautions to avoid getting solution outside of the vein.</li>
	</ol>
	</li>
	<li>Start the acquisition of the Z-stack time-lapse imaging by clicking on the Z-Stack and Time-Lapse buttons to depress them and then clicking on the record button.</li>
	<li>If other fluorescent dextrans, i.e., 10 kDa dextran-fluorescein isothiocyanate (FITC), or proteins, i.e., VEGFA165, have been prepared in advance, inject them after the start of image acquisition for a t = 0 min start.
	<ol>
		<li>Remove the syringe with PBS in the tail vein catheter and replace with the syringe containing 10 kDa dextran-FITC or VEGFA165 taking care not to introduce any bubbles into the line.</li>
		<li>Slowly inject injectables into the mouse through the tail vein catheter and replace the syringe with a syringe containing PBS.</li>
	</ol>
	</li>
	<li>Every 30-45 min, slowly inject 50 &#181;l of PBS or saline to maintain hydration of the animal.</li>
</ol>
8. Euthanasia
<ol>
	<li>At the termination of image acquisition, euthanize the animal.
	<ol>
		<li>Increase the isoflurane to 5%.</li>
		<li>Keep the animal under 5% isoflurane until 30 sec after it ceases to breathe and remove the animal from the stage.</li>
		<li>Perform cervical dislocation to ensure complete euthanasia.</li>
	</ol>
	</li>
</ol>
9. Image Processing
<ol>
	<li>Acquire images at 16-bit TIFF files for each individual channel at each time point and save sequentially.</li>
	<li>Perform separation of spectral overlap (i.e., GFP and CFP), elimination of x-y drift and the generation of movies from time-lapse sequences using established methods in ImageJ9. Perform 3D surface reconstructions of high-resolution images13.</li>
</ol>

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The authors have nothing to disclose.

Cellular interactions that occur spontaneously in the tumor microenvironment can lead to changes in tumor cell motility and intravasation. High-resolution intravital imaging of live tumor tissue permits the visualization of multi-cellular dynamics that can be highly transient10,13,24. End-point in vivo assays or time-lapse images acquired with discrete time points can provide essential information on molecular mechanisms of processes in the tumor microenvironment. Intravital imaging studies have been used to study processes in the tumor microenvironment that occur over several days, generating new insights on angiogenesis, tumor cell migration and response to drug treatment25-30. However, these assays might not capture the kinetics of transient events in the tumor microenvironment due to the time scale of the processes being studied7,31. Distinguishing multiple transient events from constant processes is a significant advantage of intravital microscopy.
The intravital imaging technique described in this protocol enables the direct visualization of cellular dynamics in the tumor microenvironment by fluorescence labeling of multiple cell lineages and structures. Multiphoton microscopy permits greater depth of imaging over single-photon confocal microscopy with depths of 300 &#181;m routinely performed24. Acquisition of Z-stacks of 50 - 100 &#181;m with 2 &#181;m steps is of high enough resolution to generate 3D reconstruction of cellular interactions, including protrusions that cells make as they extend towards each other13 and vascular structures in the tumor microenvironment. Movies and 3D reconstructions from time-lapse image sequences allow for the quantitation of cellular motility (speed and directionality) and, using changes in fluorescent signal intensity of injectable dyes, vascular permeability.
In order to capture spontaneous cellular events the number of individual images captured in a single sequence must be optimized to allow for a rate of image acquisition that captures the kinetics of the event, but that also covers a sufficient physical space to increase the probability of capturing any individual event during a single time-lapse sequence. The spatial imaging parameters that need to be considered include; the number of slices in a Z-stack, the number of fields of view with Z-stacks acquired and the axial distance between individual slices in a Z-stack. Therefore, setting these parameters before starting image acquisition is a critical step in the protocol. Using the multiphoton microscope, acquisition of 30 frames (500 &#181;m x 500 &#181;m) in a z-series is approximately 60 sec. Since the duration of observed tumor cell intravasation can be on the order of minutes13, capturing several Z-stacks for 3D reconstruction of intravasation, the acquisition time of a Z-stack was limited to 60 sec. Therefore, a single field of view with up to 30 slices in a Z-stack was acquired. Furthermore, since 3D reconstruction of cells was desired, the axial step between Z-slices was set at 2 &#181;m. This allows for sufficient information on cell volumes to allow for 3D reconstruction. These parameters must be optimized for any microscope and imaging software used based on the kinetics of the live events being studied.
The modification of the rubber pad on the microscope stage and the tail vein i.v. catheter have both allowed for a significant increase in imaging time. The rubber pad forms a well to allow for maintained hydration of the tumor while minimizing XY drift on the glass coverslip. The combination of the heating chamber with the administration of PBS maintains hydration and physiological temperature of animals. These conditions combined maintain vascular perfusion and blood flow to allow for extended imaging of cellular events at the tumor vasculature including vascular permeability and tumor cell intravasation.
A limitation of this technique is the number of fluorescent labels that can be used to visualize cells in the tumor microenvironment. Generating transgenic mice with additional cell types (i.e., endothelial cells, fibroblasts or pericytes) containing fluorescent protein can be difficult and time consuming. However, future potential applications could utilize a combination of fluorescent protein labeling of stromal cell types with implanted tumor cells and 70 kDa dextran labeling of macrophages. By using a combination of these labeling strategies and crossing transgenic mice new combinations of cellular labels can be generated to assess cellular movement and events in the tumor microenvironment.
Unlike end-point experiments based on immunofluorescence staining, high-resolution intravital microscopy permits the visualization of spontaneous and transient events in the tumor microenvironment. The intravital imaging procedure described here reveals new events and cellular interactions in the tumor microenvironment that cannot be deciphered from static images. With novel information on the interacting cells types or processes, and the kinetics of these events, hypotheses can be generated to investigate the signaling pathways that drive these interactions.

Dissemination of tumor cells from the primary mammary tumor has been shown to involve not only tumor cells, but host stromal cells including macrophages and endothelial cells. Furthermore, tumor vasculature is abnormal with increased permeability1. Thus, determining how tumor cells, macrophages and endothelial cells interact to mediate vascular permeability and tumor cell intravasation in the primary tumor microenvironment is important for understanding metastasis. Understanding the kinetics of vascular permeability, tumor cell intravasation and the underlying signaling mechanism of multicellular interactions in the tumor microenvironment can provide crucial information in the development and administration of anti-cancer therapies.
The primary means of studying tumor vascular permeability in vivo has been the measurement of extravascular dyes such as Evans blue2, high molecular weight dextrans (155 kDa)3 and fluorophore or radiotracer-conjugated proteins (including albumin)4 at fixed time points after injection of the dye. Advancements in microscopy, animal models and fluorescent dyes have enabled the visualization of cellular processes and vascular permeability in live animals through intravital microscopy5.
Live animal imaging with the acquisition of static images, or short time-lapse sequences over several time points does not allow for the complete understanding of dynamic events in the tumor microenvironment6,7. Indeed, static image acquisition over the course of 24 hr demonstrated that tumor vasculature is leaky, however the dynamics of vascular permeability was not observed6. Thus, extended continuous time-lapse imaging up to 12 hours captures the kinetics of dynamic events in the tumor microenvironment.
This protocol describes the use of extended time-lapse multiphoton intravital microscopy to study dynamic multicellular events in the tumor microenvironment. Multiple cell types in the tumor microenvironment are labeled with injectable dyes or by using transgenic animals expressing fluorescent proteins. Using a tail vein catheter vascular dyes or proteins can be injected after the start of imaging to acquire kinetic data of multicellular events in the tumor microenvironment. For live cell imaging the mammary tumor is exposed through the surgical preparation of a skin flap. Images are acquired for up to 12 hours using a multiphoton microscope equipped with multiple photomultiplier tubes (PMT) detectors8. By using appropriate filters, a subtraction algorithm enables 4 PMT detectors to acquire 5 fluorescent signals in the tumor microenvironment simultaneously9. High-resolution multiphoton intravital microscopy captures single cell resolution imaging of tumor-stroma interactions in the tumor microenvironment, leading to a better understanding of vascular permeability and tumor cell intravasation10-13. Specifically, extended intravital imaging revealed highly localized, transient vascular permeability events that occur selectively at sites of interaction between a tumor cell, a macrophage and an endothelial cell (defined as the Tumor MicroEnvironment of Metastasis, TMEM14)13. Furthermore, tumor cell intravasation occurs only at TMEM and is spatially and temporally correlated with vascular permeability13. Single cell resolution of the dynamics of these events was made possible through the use of extended time-lapse multiphoton microscopy of fluorescently labeled cells in the tumor microenvironment.

<table border="0" cellpadding="0" cellspacing="0" width="637">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:248px;">155 kDa dextran-tetramethylrhodamine isothiocyanate</td>
			<td style="width:95px;">Sigma Aldrich</td>
			<td style="width:128px;">T1287</td>
			<td style="width:167px;">reconstitute at 20 mg/mL in 1 X PBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">70 kDa dextran-Texas Red</td>
			<td style="width:95px;">Life Technologies</td>
			<td style="width:128px;">D-1830</td>
			<td>reconstitute at 10 mg/mL in 1 X PBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">10 kDa dextran-fluorescein isothyocyanate</td>
			<td style="width:95px;">Sigma Aldrich</td>
			<td style="width:128px;">FD10S</td>
			<td>reconstitute at 20 mg/mL in 1 X PBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Qdot 705 ITK Amino (PEG) Quantum Dots</td>
			<td style="width:95px;">Life Technologies</td>
			<td style="width:128px;">Q21561MP</td>
			<td>Dilute 25 uL in 175 uL of 1 X PBS for injection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">MMTV-PyMT mice</td>
			<td style="width:95px;">Jackson Laboratory</td>
			<td align="right" style="width:128px;">2374</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Csf1r-ECFP mice (Csf1r-Gal4/VP16,UAS-ECFP)</td>
			<td style="width:95px;">Jackson Laboratory</td>
			<td align="right" style="width:128px;">26051</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Csf1r-EGFP mice</td>
			<td style="width:95px;">Jackson Laboratory</td>
			<td align="right" style="width:128px;">18549</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">1 x PBS</td>
			<td style="width:95px;">Life Technologies</td>
			<td style="width:128px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:248px;">Isoethesia (isoflurane)</td>
			<td style="width:95px;">Henry Schein Animal Health</td>
			<td align="right" style="width:128px;">50033</td>
			<td>250 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Oxygen</td>
			<td style="width:95px;">AirTech</td>
			<td style="width:128px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">1 mL syringe, tuberculin slip tip</td>
			<td style="width:95px;">BD</td>
			<td align="right" style="width:128px;">309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">30G x 1 (0.3 mm X 25 mm) needle</td>
			<td style="width:95px;">BD</td>
			<td align="right" style="width:128px;">305128</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:248px;">Polyethylene micro medical tubing&#160;</td>
			<td style="width:95px;">Scientific Commodities Inc</td>
			<td style="width:128px;">BB31695-PE/1</td>
			<td>0.28 mm I.D. X 0.64 mm O.D.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Microscope coverglass</td>
			<td style="width:95px;">Corning</td>
			<td style="width:128px;">2980-225</td>
			<td>thickness 1.5, 22 X 50 mm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">MouseOx oximeter, software and sensors</td>
			<td style="width:95px;">STARR Life Sciences</td>
			<td style="width:128px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Laboratory tape</td>
			<td style="width:95px;">Fisher Scientific</td>
			<td style="width:128px;">159015R</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">soft rubber pad</td>
			<td style="width:95px;">McMaster-Carr</td>
			<td>8514K62</td>
			<td>Ultra-Soft Polyurethane Film, 3/16&#8221; Thick, 12&#34; x 12&#34;, 40 Oo Durometer, Plain Back</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">hard rubber pad</td>
			<td style="width:95px;">McMaster-Carr</td>
			<td>8568K615</td>
			<td>High-Strength Neoprene Rubber Sheet 1/4&#34; Thick, 12&#34; X 12&#34;, 50A Durometer,</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Microscope</td>
			<td style="width:95px;">Olympus</td>
			<td style="width:128px;"></td>
			<td>The microscope is a custom built two laser multiphoton microscope based on an Olympus IX-71 stand utilizing a 20x 1.05NA objective lens.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">7-Punch set</td>
			<td style="width:95px;">McMaster-Carr</td>
			<td style="width:128px;">3429A12</td>
			<td>, 1/4&#34; to 1&#34; Hole Diameter, for Hammer-Driven Hole Punch,&#160;</td>
		</tr>
	</tbody>
</table>

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.

Extended time-lapse intravital microscopy enables single cell resolution imaging of multicellular processes in the tumor microenvironment. By fluorescently labeling tumor cells, macrophages, the vascular space, and visualizing the collagen fiber network using the second harmonic generation signal, multiple compartments in the tumor microenvironment are simultaneously tracked during imaging. Tumor cells labeled with fluorescent proteins can be generated in transgenic mice as has been done in MMTV-PyMT-Dendra2 mice, with mammary tumor cells labeled with Dendra2 (Figure 1A). By crossing these transgenic mice with Csf1r-GAL4VP16/UAS-ECFP mice, which have macrophages labeled with ECFP, both tumor cells and macrophages are labeled in the MMTV-PyMT mice. With the dual labeling strategy of tumor cells and macrophages, the direct interaction of the two cells types can be visualized in real time (Figure 1A). Additionally, human breast cancer cells lines or patient-derived tumor cells transduced with GFP can be used to label tumor cells. Macrophages can be labeled using fluorescently labeled 70 kDa dextran which accumulates in the phagocytic macrophages (Figure 1B). These methods have promoted the study of dynamic multicellular events such as tumor cell-macrophage streaming motility, vascular permeability and tumor cell intravasation. To visualize changes in tumor vascular permeability, a fluorescently labeled high molecular weight dextran (155 kDa or higher is recommended9,13,21,22) or quantum dots are used to label the vascular space (Figure 1). 155 kDa dextran or quantum dots are large enough that they do not readily diffuse through the interendothelial spaces23.
Continuous high-resolution imaging facilitates the capture of vascular permeability events and the quantification of the kinetics of dextran extravasation and clearance at TMEM (Figure 2 and Movie 1). Since the vascular space is readily defined by the 155 kDa dextran at the initiation of the time-lapse sequence (as in 0&#39; of Figure 2) extravascular fluorescent dextran can be quantified in each frame of the time-lapse sequence. Figure 2 and Movie 1 demonstrate the extravasation of 155 kDa dextran-TMR injected through a tail vein catheter into a MMTV-PyMT-Dendra2; Csf1r-CFP mouse. Dendra2-labeled tumor cells (green) and CFP-labeled macrophages (cyan) are used to identify TMEM in live animals. The TMEM structure at the site of vascular permeability is outlined in a white box. The vascular space is labeled with 155 kDa dextran-TMR (red) to visualize flowing vasculature and extravasation of vascular contents during events of vascular permeability. The peak of 155 kDa dextran-TMR extravasation in Figure 2 is seen between 29&#39; and 34&#39;. Extravascular dextran is seen at the white arrowhead in the top panels and at the red arrowhead in the lower panels, showing only the channel of the 155 kDa dextran-TMR. The 155 kDa dextran-TMR clears from the tissue and is seen as a decrease in the extravascular TMR signal as seen at 97&#39; in Figure 2. After compilation of the time-lapse sequence in ImageJ extravascular dextran is quantified.
In addition to observing spontaneous events in the tumor microenvironment, it is important to study the underlying molecular mechanism of the identified phenomena. Injecting additional vascular dyes or proteins to induce vascular permeability can provide insight into the signaling events regulating vascular permeability. Figure 3 demonstrates the difference in extravasation of 10 kDa dextran-FITC (green) and 155 kDa dextran-TMR (red). High molecular weight 155 kDa dextran-TMR is administered immediately before the start of time-lapse imaging. Five Z-stacks are acquired in the time-lapse sequence to set a baseline before 10 kDa dextran-FITC is injected. After acquisition of the 5th Z-stack the 10 kDa dextran-FITC is administered through the tail vein catheter without interrupting image acquisition. The dextran-FITC is seen entering the vasculature with immediate extravasation while the 155 kDa dextran-TMR remains confined to the vascular space (Figure 3, 0&#39; and 6&#39;). The 10 kDa dextran-FITC completely extravasates from the blood vessel and clears from the tissues leaving the 155 kDa dextran-TMR in the blood vessel (Figure 3 as seen at 56&#39; and Movie 2). Using a time point from before injection, the kinetics of 10 kDa dextran-FITC extravasation can be readily compared to 155 kDa dextran-TMR13. The immediate extravasation of 10 kDa dextran-FITC after injection is significantly different from the retention of 155 kDa dextran-TMR in the vasculature and the spontaneous vascular permeability events13. Additional experimental controls can be performed including inducing vascular permeability through the mechanical disruption of the endothelium or through VEGFA165-mediated permeability13. The kinetics of extravasation of 155 kDa dextran-TMR of spontaneous permeability events can be compared to 10 kDa dextran, and other means of inducing vascular permeability to understand the events and signals involved in vascular permeability events in the tumor microenvironment.
<img alt="Figure 1" src="/files/ftp_upload/54042/54042fig1.jpg" /> 
Figure 1. Strategies for labeling cells and the vasculature in the tumor microenvironment. (A) Transgenic MMTV-PyMT mouse with tumor cells labeled with Dendra2 (MMTV-iCre/CAGCAT-Dendra2/MMTV-PyMT) and macrophages labeled with CFP (Csf1r-GAL4VP16/UAS-ECFP). Tumor vasculature is labeled with 155 kDa dextran-TMR administered by tail vein catheter. (B) TN1-GFP patient-derived xenograft tumors with macrophages labeled with 70 kDa dextran-Texas Red and vasculature labeled with quantum dots using the tail vein catheter. Scale bar, 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54042/54042fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54042/54042fig2.jpg" /> 
Figure 2. Visualizing transient vascular permeability. (A) Intravital microscopy (IVM) time-lapse of 155 kDa dextran-TMR (red) extravasation and tumor cell intravasation at TMEM (white box). Tumor cells labeled with Dendra2 (green, TC), macrophages with ECFP (cyan, M) and blood vessels with 155 kDa dextran-TMR (red, BV). Blood vessel permeability sites (white arrowhead). (B) Isolated 155 kDa dextran-TMR channel. Red arrows mark dextran extravasation (white). Scale bar, 50 &#181;m. Movie of time-lapse microscopy in Movie 1. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54042/54042fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54042/54042fig3.jpg" /> 
Figure 3. Visualizing multiple fluorescently labeled dextrans. IVM time-lapse of difference in extravasation of 155 kDa dextran-TMR (red) and 10 kDa dextran-FITC (green). Macrophages are labeled with ECFP (cyan) and collagen in blue (SHG). The overlay of red 155 kDa dextran-TMR and green 10 kDa dextran-FITC in the vasculature is yellow. Rapid extravasation of green 10 kDa dextran-FITC peak is at 6 min. Scale bar, 50 &#181;m. Movie of time-lapse microscopy in Movie 2. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54042/54042fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Movie 1" src="/files/ftp_upload/54042/54042movie1.jpg" /> 
Movie 1. Visualizing spontaneous transient vascular permeability with time-lapse IVM. Time-lapse microscopy (as seen in Figure 2) of transient blood vessel permeability visualized in a transgenic MMTV-PyMT mouse with tumor cells labeled with Dendra2 (MMTV-iCre/CAGCAT-Dendra2/MMTV-PyMT) and macrophages labeled with CFP (Csf1r-GAL4VP16/UAS-ECFP). Extravasation of 155 kDa dextran-TMR extravasation (red in left panel, white right panel) is observed at the branch point of the tumor blood vessel. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54042/Movie_1-JoVE_Harney.avi" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Movie 2" src="/files/ftp_upload/54042/54042movie2.jpg" /> 
Movie 2. Visualizing extravasation of multiple fluorescently labeled dextrans by time-lapse IVM. Time-lapse microscopy (as seen in Figure 3) of extravasation of 155 kDa dextran-TMR (red) and 10 kDa dextran-FITC (green) in a transgenic MMTV-PyMT mouse with macrophages labeled with CFP (Csf1r-GAL4VP16/UAS-ECFP). SHG of collagen is labeled in blue. Red 155 kDa dextran-TMR remains in the tumor vasculature for the duration of the time-lapse while the green 10 kDa dextran-FITC rapidly extravasates and clears from the blood vessels and the tissue. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54042/Movie_2-JoVE_Harney.avi" target="_blank">Please click here to view this video. (Right-click to download.)</a>

Watch this Scientific Journal Video about Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment at JoVE.com

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics 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 ...

extended-time-lapse-intravital-imaging-real-time-multicellular

Nanyang Technological University

Research

JoVE Journal

Medicine

8.0K Views.  Albert Einstein College of Medicine. 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.

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

Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical excision of the tumor with a full lymphadenectomy in the drainage area of the tumor. However, removal of lymph nodes is associated with increased morbidity due to infection, wound breakdown and lymphedema.4,5 As an alternative, the sentinel lymph node procedure (SLN) was developed several decades ago to detect the first draining lymph node from the tumor.6 In case of lymphogenic dissemination, the SLN is the first lymph node that is affected (Figure 1). Hence, if the SLN does not contain metastases, downstream lymph nodes will also be free from tumor metastases and need not to be removed. The SLN procedure is part of the treatment for many tumor types, like breast cancer and melanoma, but also for cancer of the vulva and cervix.7 The current standard methodology for SLN-detection is by peritumoral injection of radiocolloid one day prior to surgery, and a colored dye intraoperatively. Disadvantages of the procedure in cervical and vulvar cancer are multiple injections in the genital area, leading to increased psychological distress for the patient, and the use of radioactive colloid.
Multispectral fluorescence imaging is an emerging imaging modality that can be applied intraoperatively without the need for injection of radiocolloid. For intraoperative fluorescence imaging, two components are needed: a fluorescent agent and a quantitative optical system for intraoperative imaging. As a fluorophore we have used indocyanine green (ICG). ICG has been used for many decades to assess cardiac function, cerebral perfusion and liver perfusion.8 It is an inert drug with a safe pharmaco-biological profile. When excited at around 750 nm, it emits light in the near-infrared spectrum around 800 nm. A custom-made multispectral fluorescence imaging camera system was used.9.
The aim of this video article is to demonstrate the detection of the SLN using intraoperative fluorescence imaging in patients with cervical and vulvar cancer. Fluorescence imaging is used in conjunction with the standard procedure, consisting of radiocolloid and a blue dye. In the future, intraoperative fluorescence imaging might replace the current method and is also easily transferable to other indications like breast cancer and melanoma.

Fluorescence imaging is a promising innovative modality for image-guided surgery in surgical oncology. In this video we describe the technical procedure for detection of the sentinel lymph node using fluorescence imaging as showcased in gynecologic oncologicy. A multispectral fluorescence camera system, together with the fluorescent agent indocyanine green, is applied.

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical ...

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury (IRI) of the Rat Cremaster Muscle

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, intestinal ischemia as well as following transplant and cardiovascular surgery.1 Reperfusion of previously ischemic tissue, while essential for the prevention of irreversible tissue injury, elicits excessive inflammation of the affected tissue. Adjacent to the production of reactive oxygen species, activation of the complement system and increased microvascular permeability, the activation of leukocytes is one of the principle actors in the pathological cascade of inflammatory tissue damage during reperfusion.2, 3 Leukocyte activation is a multistep process consisting of rolling, firm adhesion and transmigration and is mediated by a complex interaction between adhesion molecules in response to chemoattractants such as complement factors, chemokines, or platelet-activating factor.4
While leukocyte rolling in postcapillary venules is predominantly mediated by the interaction of selectins5 with their counter ligands, firm adhesion of leukocytes to the endothelium is selectin-controlled via binding to intercellular adhesion molecules (ICAM) and vascular cellular adhesion molecules (VCAM).6, 7 
Gold standard for the in vivo observation of leukocyte-endothelial interaction is the technique of intravital microscopy, first described in 1968.8
Though various models of IRI (ischemia-reperfusion injury) have been described for various organs, 9-12 only few are suitable for direct visualization of leukocyte recruitment in the microvascular bed on a high level of image quality.8 
We here promote the digital intravital epifluorescence microscopy of the postcapillary venule in the cremasteric microcirculation of the rat 13 as a convenient method to qualitatively and quantitatively analyze leukocyte recruitment for IRI-research in striated muscle tissue and provide a detailed manual for accomplishing the technique. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly appreciate, and safely perform the method.
In a step by step protocol we depict how to get started with respiration controlled anesthesia under sufficient monitoring to keep the animal firmly anesthetized for longer periods of time. We then describe the cremasteric preparation as a thin flat sheet for outstanding optical resolution and provide a protocol for leukocyte imaging in IRI that has been well established in our laboratories.

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury IRI of the Rat Cremaster Muscle

Digital intravital epifluorescence microscopy of postcapillary venules in the cremasteric microcirculation is a convenient method to gain insights into leukocyte-endothelial interaction in vivo in ischemia-reperfusion injury (IRI) of striated muscle tissue. We here provide a detailed protocol to safely perform the technique and discuss its applications and limitations.

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Confocal Time Lapse Imaging as an Efficient Method for the Cytocompatibility Evaluation of Dental Composites

It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The objective of this study was to use 3D CLSM time lapse confocal imaging to assess the in vitro biocompatibility of dental composites. This method provides an accurate and sensitive indication of viable cell rate in contact with dental composite extracts. The ELS extra low shrinkage, a dental composite used for direct restoration, has been taken as example. In vitro assessment was performed on cultured primary human gingival fibroblast cells using Live/Dead staining. Images were obtained with the FV10i confocal biological inverted system and analyzed with the FV10-ASW 3.1 Software. Image analysis showed a very slight cytotoxicity in the presence of the tested composite after 5 hours of time lapse. A slight decrease of cell viability was shown in contact with the tested composite extracts compared to control cells. The findings highlighted the use of 3D CLSM time lapse imaging as a sensitive method to qualitatively and quantitatively evaluate the biocompatibility behavior of dental composites.

The most studied aspect of the biocompatibility of dental composites is their cytotoxicity 3D CLSM time lapse imaging combined with fluorescent signal quantification allows sensitive evaluation of the cell fate over time. Utilizing this method could be an efficient tool to assess the cytocompatibility of dental composites.

It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The ...

Real-time X-ray Imaging of Lung Fluid Volumes in Neonatal Mouse Lung

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting and sustaining this phenotype is critically important in normal alveolar growth and gas exchange throughout life. Several in vitro studies have characterized the role of key regulatory proteins, signaling molecules, and steroid hormones that can influence the rate of lung fluid clearance. However, in vivo examinations must be performed to evaluate whether these regulatory factors play important physiological roles in regulating perinatal lung liquid absorption. As such, the utilization of real time X-ray imaging to determine perinatal lung fluid clearance, or pulmonary edema, represents a technological advancement in the field. Herein, we explain and illustrate an approach to assess the rate of alveolar lung fluid clearance and alveolar flooding in C57BL/6 mice at post natal day 10 using X-ray imaging and analysis. Successful implementation of this protocol requires prior approval from institutional animal care and use committees (IACUC), an in vivo small animal X-ray imaging system, and compatible molecular imaging software.

We present a protocol to assess the rate of alveolar fluid clearance or pulmonary edema in neonatal mouse lung using X-ray imaging technology.

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

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

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Chapters in this video

Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury (IRI) of the Rat Cremaster Muscle

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Confocal Time Lapse Imaging as an Efficient Method for the Cytocompatibility Evaluation of Dental Composites

Real-time X-ray Imaging of Lung Fluid Volumes in Neonatal Mouse Lung

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR