Name: long-term high-resolution intravital microscopy in the lung with a vacuum stabilized imaging window
Uploaded: 2016-10-06T15:00:00
Description: Watch this Scientific Journal Video about Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window at JoVE.com

This research was supported by NIH-CA100324, Einstein National Cancer Institute's cancer center support grant P30CA013330, R01CA172451 to JWP and the Integrated Imaging Program. This technology was developed in the Gruss-Lipper Biophotonics Center and the Integrated Imaging Program at the Albert Einstein College of Medicine. We acknowledge the support of these Centers in this work. The authors thank Mike Rottenkolber, Ricardo Ibagon and Anthony Leggiadro of the Einstein machine shop for their skilled and timely craftsmanship, the laboratory of Matthew Krummel for generously sharing their window design drawings, Kevin Elicieri and Jeremy Bredfeldt for their expertise in microscopy and their amplifier recommendations and Allison Harney and Bojana Gligorijevic for informative discussions.

All procedures described in this protocol have been 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 Mouse Model and Tumor Cells
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	<li>Prepare 100 ml of 0.1 % (w/v) bovine serum albumin/phosphate buffered saline (BSA/PBS) buffer by mixing 0.1 g of BSA with 100 ml of PBS.</li>
	<li>Prepare fluorescent...

High resolution in vivo optical imaging combined with fluorescently labelled functional tags such as proteins and antibodies has dramatically increased our understanding of the metastatic cascade. It has enabled direct visualization and quantification of single-cell and sub-cellular parameters in tumor cells, host cells and their microenvironment. This imaging within the primary tumor has led, for example, to the discovery of discrete microenvironments that are supportive of either growth invasion or disseminati...

High resolution intravital optical imaging has proven to be crucial to understanding many biological processes, allowing single-cell and sub-cellular parameters to be measured and quantified. In cancer research, intravital imaging of tumor and stromal cells has led to the discovery of many microenvironmental interactions 6-11 that are only present in the intact animal.
Discoveries about microenvironments associated with intravasation and dissemination of tumor cells in breast cancer using single cell resolution optical imaging in vivo has even led to novel markers for prognosis and response to treatment in breast cancer patients 12-16. The best imaging technologies available for viewing deep within intact internal vital organs are the clinical modalities (MRI, PET, CT) which offer excellent views of the entire organ and can reveal pathologies even before they produce clinical symptoms. They are unable, however, to reveal the earliest stages of metastasis and the cellular mechanisms driving tumor progression due to their lack of single cell resolution. By the time lung metastases are visible in these modalities, they are well established and proliferating. Given the estimate that 90% of disseminated tumor cells that arrive to the lung either do not survive 17 or initially remain dormant 18, and the observation that they arrive much earlier than previously expected 19, imaging the earliest steps of arrival and survival becomes crucial to understanding the process of metastatic seeding and recurrence of tumor growth at distant sites.
Performing these observations in the lung has proven extremely difficult however; the vast majority of imaging studies have utilized ex vivo or explant preparations 20-23, which only give a view into the lung at single time points. While these preparations do provide useful information, they do not give a complete understanding of the interactions, cause and effect relationships, and dynamics that occur between the various components of the microenvironment. The lack of a proper circulatory system (and concomitant imbalance of homeostasis) and the disconnection from the rest of the body&#39;s immune system makes it desirous to validate the conclusions that these preparations generate in intact tissue in vivo.
Many groups have performed intravital imaging of the intact lung 2,4,5,24-33 with Wearn and German being the first to surgically expose the pleural layer 24 and Terry the first to utilize an implantable imaging window 25.
High resolution imaging in the lung is greatly hindered by the lung&#39;s constant motion and several techniques have been developed to overcome this limitation. Wagner and Filley 27 studied the natural motion of the canine lung and designed their surgical protocol to locate their implanted window over a relatively stationary region while Wagner utilized vacuum in his window surgical preparation to immobilize the tissue 28. Since that time, a variety of techniques have been utilized to image the lung including: bronchus clamping, sequential apnea and gated imaging, oversampled acquisition, gluing of the lung lobe and vacuum 34. Each of these has its advantages and disadvantages and no one technique has emerged as superior to another 34. For example, bronchus clamping and sequential apnea alter the normal exchange of gases in the lung and may cause atelectasis. Gated imaging and oversampled acquisition do not suffer from these disadvantages but require high-speed or specialized imaging equipment not widely accessible. Finally both gluing of the lung and the vacuum technique avoid both of the above mentioned drawbacks, but may exhibit shear force induced injury if care is not taken. In recent years, the vacuum window has been miniaturized and adapted for use in mice using confocal and multiphoton microscopy 4,5,33 and excellent high-resolution imaging has been attained 2. Table 1 summarizes this rich history and highlights those papers which describe novel advancements in the use of intravital lung imaging windows.
This protocol describes the use of extended time-lapse multiphoton intravital microscopy to image metastasis in the live, intact lung with the highest subcellular resolution possible. Images are acquired for up to 12 hr using a multiphoton microscope equipped with a high numerical aperture objective lens and multiple photomultiplier tube (PMT) detectors. Transgenic mouse models are utilized to fluorescently label native macrophages along with fluorescent high molecular weight dextran and fluorescent protein transfected tumor cells (to label the vasculature and tumor cells respectively). While this choice of fluorescently labeled cells enables visualization of tumor cell-endothelial cell-macrophage interactions and dynamics, this protocol will work for any strain of fluorescent or non-fluorescent mouse. After acquisition, residual drift motion (if any) is eliminated using a Fiji plugin 35,36 and custom macros time average the vascular channel to eliminate flashing caused by unlabeled circulating blood cells.
While this protocol focuses on imaging metastasis, the techniques are applicable to many other biological processes observable with high-resolution single-cell imaging in the lung.

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			<td height="21" style="height:21px;width:924px;">Nickel-Plated Brass Vacuum Regulator 1/8 NPT Female, w/ Gauge, 0 - 20&#34; Hg Vacuum</td>
			<td style="width:291px;">McMaster Carr</td>
			<td style="width:141px;">4172K12&#160;</td>
			<td style="width:223px;">Vacuum Regulator</td>
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			<td height="21" style="height:21px;width:924px;">Brass Barbed Hose Fitting Adapter for 1/4&#34; Hose ID X 1/8&#34; NPTF Male Pipe</td>
			<td style="width:291px;">McMaster Carr</td>
			<td style="width:141px;">5346K13</td>
			<td>Vacuum Regulator Hose Adapter</td>
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			<td height="21" style="height:21px;width:924px;">Pyrex Brand Filtering Flasks with Tubulation; Neck tooled for rubber stopper No. 4; Capacity: 50mL</td>
			<td style="width:291px;">Corning Life Sciences Glass</td>
			<td style="width:141px;">5360-50</td>
			<td>Vacuum Flask</td>
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			<td height="21" style="height:21px;width:924px;">Round Glass Coverslips Thickness #1.5, 0.16-0.19mm 10mm dia.&#160;</td>
			<td style="width:291px;">Ted Pella, Inc.</td>
			<td>260368</td>
			<td>Cover slips</td>
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			<td height="21" style="height:21px;width:924px;">Exel International Disposable Safelet I.V. Catheters; 22gx1 in.&#160;</td>
			<td style="width:291px;">Exel International</td>
			<td style="width:141px;">26746</td>
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			<td height="21" style="height:21px;width:924px;">PERMA-HAND Black Braided Silk Sutures, ETHICON LIGAPAK Dispensing Reel Size 2-0</td>
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			<td style="width:141px;">95056-992</td>
			<td>String</td>
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			<td height="21" style="height:21px;width:924px;">Liquid Super Glue, Clear, 0.14oz</td>
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			<td>Cyano-acrylate Glue</td>
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			<td height="21" style="height:21px;width:924px;">Tetramethylrhodamine isothiocyanate&#8211;Dextran</td>
			<td style="width:291px;">Sigma-Aldrich</td>
			<td style="width:141px;">T1287-500MG</td>
			<td>155kD Dextran</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Laboratory Clear Tygon PVC Tubing, 1/16&#34; ID, 1/8&#34; OD, 1/32&#34; Wall Thickness, 25 ft. Length</td>
			<td style="width:291px;">McMaster Carr</td>
			<td style="width:141px;">5155T12</td>
			<td>Thin Tubing &#38; Tubing for Luer</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Crack-Resistant Polyethylene Tubing, 1/8&#34; ID, 1/4&#34; OD, 1/16&#34; Wall Thickness, White, 50 ft. Length&#160;</td>
			<td style="width:291px;">McMaster Carr</td>
			<td style="width:141px;">5181K24&#160;</td>
			<td>Thick Tubing</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Depillatory Lotion</td>
			<td style="width:291px;">Nair</td>
			<td style="width:141px;">-</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Micro Medical Tubing 95 Durometer LDPE</td>
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			<td style="width:141px;">BB31695-PE/1</td>
			<td>Tubing for tail vein catheter</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:924px;">30 G x 1 in. BD PrecisionGlide Needle</td>
			<td style="width:291px;">BD</td>
			<td style="width:141px;">305128</td>
			<td>Needles for tail vein catheter</td>
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			<td height="21" style="height:21px;width:924px;">Puritan Nonsterile Cotton-Tipped Swabs&#160;</td>
			<td style="width:291px;">Fisher Scientific</td>
			<td>867WCNOGLUE</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Clear Polycarbonate Barbed Tube Fitting, Reducing Straight for 3/32&#34; x 1/16&#34; Tube ID</td>
			<td style="width:291px;">McMaster Carr</td>
			<td style="width:141px;">5117k51</td>
			<td>Connectors between tubes</td>
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			<td height="21" style="height:21px;width:924px;">One-Hole Rubber Stoppers</td>
			<td style="width:291px;">Fisher Scientific</td>
			<td>14-135F</td>
			<td>Stopper for Vacuum Flask</td>
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			<td height="21" style="height:21px;width:924px;">SHARP Precision Barrier Tips, For P-100, 100&#181;l</td>
			<td style="width:291px;">Denville Scientific Inc.</td>
			<td style="width:141px;">P1125</td>
			<td>Pipette Tip</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Laboratory tape</td>
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			<td style="width:141px;">159015R</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Puralube</td>
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			<td style="width:141px;">008897</td>
			<td>Opthalmic Ointment</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Gemini Cautery Kit</td>
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			<td style="width:141px;">726067</td>
			<td>Cautery Pen</td>
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			<td height="21" style="height:21px;width:924px;">Graefe Micro Dissecting Forceps; Serrated; Slight Curve; 0.8mm Tip Width; 4&#34; Length</td>
			<td style="width:291px;">Roboz Surgical</td>
			<td style="width:141px;">RS-5135&#160;</td>
			<td>Forceps</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Extra Fine Micro Dissecting Scissors 4&#34; Straight Sharp/Sharp 24mm</td>
			<td style="width:291px;">Roboz Surgical</td>
			<td style="width:141px;">RS-5912</td>
			<td>Sharp Scissors</td>
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			<td height="21" style="height:21px;width:924px;">Micro Dissecting Scissors 4&#34; Straight Blunt/Blunt</td>
			<td style="width:291px;">Roboz Surgical</td>
			<td style="width:141px;">RS-5980</td>
			<td>Blunt Scissors</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Wipes</td>
			<td style="width:291px;">Fisher Scientific</td>
			<td style="width:141px;">06-666-A&#160;</td>
			<td>Harness</td>
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			<td height="21" style="height:21px;width:924px;">PhysioSuite System</td>
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			<td>Vitals Monitor</td>
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			<td height="21" style="height:21px;width:924px;">1 mL Syringe, Tuberculin Slip Tip</td>
			<td style="width:291px;">BD</td>
			<td style="width:141px;">309659</td>
			<td>Syringe</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Cyano acrylate</td>
			<td style="width:291px;">Staples</td>
			<td>LOC1647358</td>
			<td>Cover Slip Adhesive</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Petroleum Jelly</td>
			<td style="width:291px;">Fisher Scientific</td>
			<td style="width:141px;">19-086291</td>
			<td>Water Barrier</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Adapter Luer Cannulla 1.5-2.2mm</td>
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			<td style="width:141px;">734118</td>
			<td>Catheter Connector</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:924px;">MouseOx oximeter, software and sensors</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Isoethesia (isoflurane)</td>
			<td style="width:291px;">Henry Schein Animal Health</td>
			<td style="width:141px;">50033</td>
			<td>250 mL</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Oxygen</td>
			<td style="width:291px;">TechAir</td>
			<td style="width:141px;">OX TM</td>
			<td></td>
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			<td height="21" style="height:21px;width:924px;">1 x PBS</td>
			<td style="width:291px;">Life Technologies</td>
			<td>10010-023</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:924px;">PVC Ball Valve, Push to Connect, 1/4 In</td>
			<td style="width:291px;">Grainger</td>
			<td>3CGJ7</td>
			<td>Vacuum Valve</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Small Animal Ventilator</td>
			<td style="width:291px;">Harvard Apparatus</td>
			<td style="width:141px;">683</td>
			<td>Alternative is available from Kent Scientific: MouseVent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">OptiMEM Reduced Serum Medium</td>
			<td style="width:291px;">ThermoFisher Scientific</td>
			<td style="width:141px;">31985062&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Lipofectamine 2000 Transfection Reagent</td>
			<td style="width:291px;">ThermoFisher Scientific</td>
			<td style="width:141px;">11668019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">MacBlue Tg(Csf1r*-GAL4/VP16,UAS-ECFP)1Hume/J Mice</td>
			<td style="width:291px;">Jackson Laboratory</td>
			<td style="width:141px;">026051&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Multiphoton Microscope</td>
			<td style="width:291px;">Olympus</td>
			<td style="width:141px;">Fluoview FV1000</td>
			<td>Alternative to custom built scope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Environmental Enclosure</td>
			<td style="width:291px;">Precision Plastics</td>
			<td style="width:141px;">Chamber for FV1000</td>
			<td>Alternative to custom built enclosure</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Phosphate Buffered Saline</td>
			<td style="width:291px;">ThermoFisher Scientific</td>
			<td>14190136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Laser Power Meter</td>
			<td style="width:291px;">Coherent</td>
			<td style="width:141px;">FieldMaxIITOP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Laser Power Meter Head</td>
			<td style="width:291px;">Coherent</td>
			<td style="width:141px;">PM10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">pcDNA3-Clover Fluorescent Protein Vector</td>
			<td style="width:291px;">Addgene</td>
			<td style="width:141px;">40259</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">G418 Sulfate Selective Antibiotic</td>
			<td style="width:291px;">ThermoFisher Scientific</td>
			<td style="width:141px;">10131027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">MoFlo Fluorescent-Activate Cell Sorter&#160;</td>
			<td style="width:291px;">Beckman Coulter</td>
			<td style="width:141px;">XDP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Trypsin EDTA 1X</td>
			<td style="width:291px;">Corning</td>
			<td style="width:141px;">25-052-Cl</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">40 &#181;m Mesh</td>
			<td style="width:291px;">Falcon</td>
			<td style="width:141px;">352235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">96 Well Plate</td>
			<td style="width:291px;">Costar</td>
			<td style="width:141px;">3599</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">60 mm Culture Dish</td>
			<td style="width:291px;">Corning</td>
			<td style="width:141px;">430196</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">10 cm Culture Dish</td>
			<td style="width:291px;">Corning</td>
			<td style="width:141px;">353003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Bovine Serum Albumin</td>
			<td style="width:291px;">Sigma-Aldrich</td>
			<td style="width:141px;">A4503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Dulbecco&#39;s Phosphate Buffered Saline 1X</td>
			<td style="width:291px;">Corning</td>
			<td style="width:141px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">C57BL/6J Mouse</td>
			<td style="width:291px;">Jackson Laboratory</td>
			<td style="width:141px;">000664&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:924px;">Kim Wipes</td>
			<td style="width:291px;">Fisher Scientific</td>
			<td style="width:141px;">06-666-A&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

long-term high-resolution intravital microscopy in the lung with a vacuum stabilized imaging window

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

To demonstrate the type of results that can be achieved with this method, we injected E0771-LG tumor cells labeled with the fluorescent protein Clover into the tail vein of MacBlue mice 44 at varying time points before surgery. After surgery, 155 kD rhodamine labeled dextran was injected IV to mark the vasculature and time-lapse imaging was performed.
When imaging mice 24 hr post injection, single cells are visible in...

Watch this Scientific Journal Video about Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window at JoVE.com

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

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

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

The overall goal of this method is to perform long-term, high-resolution, single-cell imaging of the intact lung using a vacuum stabilized imaging window. This method can help answer key questions in the field of cancer research;such as, what interactions secure between stroma and tumor cells during the earliest steps of metastasis. The main advantage of this technique is that the live, intact lung can be theorically visualized at a single-cell resolution for up to 12 hours.Begin by tying a three inch length of 2-0 suture to a tracheal catheter, one quarter inch above the bushing, with a double-knot. Then apply eye ointment to an anesthetized mouse's eyes and use depilatory cream to remove the hair from the left side of the animal from the mid-line of the chest to a quarter of the back and from the axilla to just under the rib cage. Clean any excess lotion and sterilize the exposed skin with 70%ethanol.Next, intubate the animal. And tie the 2-0 suture around the snout, under the front teeth. Move the animal to the surgical area, taking care not to dislodge the catheters, and turn on the ventilator.Connect the ventilator to the tracheal catheter. Then tape the catheter to the snout and the left forelimb to the catheter. After confirming a lack of response to toe-pinch, use sharp scissors to remove one square centimeter of skin above the left chest wall.Lift the mammary fat pad and cauterize any exposed blood vessels. Next, resect the fat pad and remove the muscle layer down to the rib cage, taking care not to cut the axillary vein at the base of the forelimb. Use forceps to lift the sixth rib.Then, holding the sharp scissors at a shallow five degree angle, cut the rib near the edge of the opening in the skin. Remove four consecutive ribs to widen the opening in the chest wall, exposing the entire lung lobe. Then carefully transfer the mouse onto the microscope imaging stage.With the vacuum off, fill the inner chamber of the vacuum window with PBS. Invert the mouse and position the exposed lung over the vacuum imaging coverslip window. Slowly, turn on the vacuum.Place a restraining harness, made of tissue paper folded in half, twice over the chest of the mouse and tape the harness to the stage plate. Then attach a pulse oximeter to the animal's paw. Then place the environmental chamber on the stage and turn on the heat to maintain the mouse at a physiological temperature.When the animal is in position, bring the 25 by 95 numerical aperture objective lens near the cover slip. In the epifluorescence mode, view the fitzy channel and bring the lung tissue into focus. Next, load a sterile syringe with 100 microliters of the tumor cell suspension, remove the syringe of PBS from the tail vein catheter, and connect the tumor cell syringe.Slowly inject the tumor cells into the tail vein. Then reconnect the PBS syringe to the tail vein catheter and locate the tumor cells throughout the lung tissue via the microscope ocular. Now replace the PBS syringe with a syringe loaded with 100 microliters of rhodamine dextran and slowly inject the dextran into the mouse via the tail catheter.Follow the dextran administration with a 50 microliter, sterile, PBS flush. Then obtain images at each of the previously located tumor cell locations to visualize the flow and integrity of the vasculature. Twenty-four hours post injection, single tumor cells can be visualized interacting with macrophages and monocytes within the vasculature.The stability of the imaging is such that sequential Z-stacks of the field can be acquired and a three dimensional reconstruction can be generated. Here, a five by five mosaic of imaging fields, covering an 890 micron field of view, over 200 and five minutes, at 15 microns below the surface of the lung of a single metastatic lesion, 12 days post injection is shown. Despite the large field of view, the high resolution of the underlying frames, composing the mosaic, enables the capture of subcellular events, such as the mitosis of a single cell, as evidenced by chromosomal separation.Intravascular injection of a high molecular weight, fluorescently labeled dextran, results in the labeling of the vascular lumen;although, unlabeled circulating erythrocytes and leukocytes can occlude the dextran. In the small capillaries of the lung, this occlusion results in a flashing of the dextran signal and a loss of definition in the vascular boundaries. The high spatial stability, provided by this protocol, allows time-averaging of the blood channel without blurring, thus restoring the temporary occlusions.The other signal channels can then be overlayed on the vasculature to provide a clear view of the vessel boundaries. Once mastered, this technique can be completed in about an hour if it's performed properly. While attempting this procedure, it's important to remember to take care not to cut any large blood vessels without first cauterizing them and to avoid touching the lung tissue at all.Following this procedure, the earliest stages of tumor cell dissemination and proliferation can be visualized. After watching this video, you should have a good understanding of how to perform intravital imaging in the intact lung with a vacuum stabilized imaging window. Thanks for watching and good luck with your experiment.

long-term-high-resolution-intravital-microscopy-lung-with-vacuum

Research

JoVE Journal

Cancer Research

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over the past decade, new and innovative technologies have greatly enhanced the practicality of live-cell imaging. Cells can now be kept in focus and continuously imaged over several days while maintained under 37 &#176;C and 5% CO2 cell culture conditions. Moreover, multiple fields of view representing different experimental conditions can be acquired simultaneously, thus providing high-throughput experimental data. Live-cell imaging provides a significant advantage over fixed-cell imaging by allowing for the direct visualization and temporal quantitation of dynamic cellular events. Live-cell imaging can also identify variation in the behavior of single cells that would otherwise have been missed using population-based assays. Here, we describe live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel. We demonstrate methods to visualize whether mitotically arrested cells die directly from mitosis or slip back into interphase. We also describe how the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system can be used to assess the fraction of interphase cells born from mitotic slippage that are capable of re-entering the cell cycle. Finally, we describe a live-cell imaging method to identify nuclear envelope rupture events.

Live-cell imaging provides a wealth of information on single cells or whole populations that is unattainable by fixed cell imaging alone. Here, live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel are described.

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over ...

Characterization of the Effects of Migrastatic Inhibitors on 3D Tumor Spheroid Invasion by High-resolution Confocal Microscopy

Drug discovery and development in cancer research is increasingly being based on drug screens in a 3D format. Novel inhibitors targeting the migratory and invasive potential of cancer cells, and consequently the metastatic spread of disease, are being discovered and considered as complementary treatments in highly invasive cancers such as gliomas. Thus, generating data enabling the detailed analyses of cells in a 3D environment following the addition of a drug is required. The methodology described here, combining spheroid invasion assays with high-resolution image capture and data analysis by confocal laser scanning microscopy (CLSM), enabled detailed characterization of the effects of the potential anti-migratory inhibitor MI-192 on glioma cells. Spheroids were generated from cell lines for invasion assays in low adherent 96-well plates and then prepared for CLSM analysis. The described workflow was preferred over other commonly used spheroid-generating techniques due to both ease and reproducibility. This, combined with the enhanced image resolution attained by confocal microscopy compared to conventional wide-field approaches, allowed the identification and analysis of distinct morphological changes in migratory cells in a 3D environment following treatment with the migrastatic drug MI-192.

The effects of migrastatic inhibitors on glioma cancer cell migration in three-dimensional (3D) invasion assays using a histone deacetylase (HDAC) inhibitor are characterized by high-resolution confocal microscopy.

Drug discovery and development in cancer research is increasingly being based on drug screens in a 3D format. Novel inhibitors targeting the migratory and ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are particularly devastating, difficult to treat, and confer a poor prognosis. While the precise incidence of brain metastases in the United States remains hard to estimate, it is likely to increase as extracranial therapies continue to become more efficacious in treating cancer. Thus, it is necessary to identify and develop novel therapeutic approaches to treat metastasis at this site. To this end, intracranial injection of cancer cells has become a well-established method in which to model brain metastasis. Previously, the inability to directly measure tumor growth has been a technical hindrance to this model; however, increasing availability and quality of small animal imaging modalities, such as magnetic resonance imaging (MRI), are vastly improving the ability to monitor tumor growth over time and infer changes within the brain during the experimental period. Herein, intracranial injection of murine mammary tumor cells into immunocompetent mice followed by MRI is demonstrated. The presented injection approach utilizes isoflurane anesthesia and a stereotactic setup with a digitally controlled, automated drill and needle injection to enhance precision, and reduce technical error. MRI is measured over time using a 9.4 Tesla instrument in The Ohio State University James Comprehensive Cancer Center Small Animal Imaging Shared Resource. Tumor volume measurements are demonstrated at each time point through use of ImageJ. Overall, this intracranial injection approach allows for precise injection, day-to-day monitoring, and accurate tumor volume measurements, which combined greatly enhance the utility of this model system to test novel hypotheses on the drivers of brain metastases.

Intracranial brain metastasis modeling is complicated by an inability to monitor tumor size and response to treatment with precise and timely methods. The presented methodology couples intracranial tumor injection with magnetic resonance imaging analysis, which when combined, cultivates precise and consistent injections, enhanced animal monitoring, and accurate tumor volume measurements.

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...

Longitudinal Intravital Imaging Through Clear Silicone Windows

Intravital microscopy (IVM) enables visualization of cell movement, division, and death at single-cell resolution. IVM through surgically inserted imaging windows is particularly powerful because it allows longitudinal observation of the same tissue over days to weeks. Typical imaging windows comprise a glass coverslip in a biocompatible metal frame sutured to the mouse&#8217;s skin. These windows can interfere with the free movement of the mice, elicit a strong inflammatory response, and fail due to broken glass or torn sutures, any of which may necessitate euthanasia. To address these issues, windows for long-term abdominal organ and mammary gland imaging&#160;were developed from a thin film of polydimethylsiloxane (PDMS), an optically clear silicone polymer previously used for cranial imaging windows. These windows can be glued directly to the tissues, reducing the time needed for insertion. PDMS is flexible, contributing to its durability in mice over time&#8212;up to 35 days have been tested. Longitudinal imaging is imaging of the same tissue region during separate sessions. A stainless-steel grid was embedded within the windows to localize the same region, allowing the visualization of dynamic processes (like mammary gland involution) at the same locations, days apart. This silicone window also allowed monitoring of single disseminated cancer cells developing into micro-metastases over time. The silicone windows used in this study are simpler to insert than metal-framed glass windows and cause limited inflammation of the imaged tissues. Moreover, embedded grids allow for straightforward tracking of the same tissue region in repeated imaging sessions.

An approach is here presented for long-term intravital imaging using optically clear, silicone windows that can be glued directly to the tissue/organ of interest and the skin.&#160;These windows are cheaper and more versatile than others currently used in the field, and the surgical insertion causes limited inflammation and distress to the animals.

Intravital microscopy (IVM) enables visualization of cell movement, division, and death at single-cell resolution. IVM through surgically inserted imaging ...

A Permanent Window for Investigating Cancer Metastasis to the Lung

Metastasis, accounting for ~90% of cancer-related mortality, involves the systemic spread of cancer cells from primary tumors to secondary sites such as the bone, brain, and lung. Although extensively studied, the mechanistic details of this process remain poorly understood. While common imaging modalities, including computed tomography (CT), positron emission tomography (PET), and magnetic resonance imaging (MRI), offer varying degrees of gross visualization, each lacks the temporal and spatial resolution necessary to detect the dynamics of individual tumor cells. To address this, numerous techniques have been described for intravital imaging of common metastatic sites. Of these sites, the lung has proven especially challenging to access for intravital imaging owing to its delicacy and critical role in sustaining life. Although several approaches have previously been described for single-cell intravital imaging of the intact lung, all involve highly invasive and terminal procedures, limiting the maximum possible imaging duration to 6-12 h. Described here is an improved technique for the permanent implantation of a minimally invasive thoracic optical Window for High-Resolution Imaging of the Lung (WHRIL). Combined with an adapted approach to microcartography, the innovative optical window facilitates serial intravital imaging of the intact lung at single-cell resolution across multiple imaging sessions and spanning multiple weeks. Given the unprecedented duration of time over which imaging data can be gathered, the WHRIL can facilitate the accelerated discovery of the dynamic mechanisms underlying metastatic progression and numerous additional biologic processes within the lung.

Here, we present a protocol for the surgical implantation of a permanently indwelling optical window for the murine thorax, which enables high-resolution, intravital imaging of the lung. The permanence of the window makes it well-suited to the study of dynamic cellular processes in the lung, especially those that are slowly evolving, such as metastatic progression of disseminated tumor cells.

Metastasis, accounting for ~90% of cancer-related mortality, involves the systemic spread of cancer cells from primary tumors to secondary sites such as ...

High-Resolution Ultrasonography for the Analysis of Orthotopic ATC Tumors in a Genetically Engineered Mouse Model

Anaplastic thyroid carcinoma (ATC) is associated with a poor prognosis and short median survival time, but no effective treatment improves the outcomes significantly. Genetically engineered murine models that mimic ATC&#39;s progression may help researchers to study treatments for this disease. Crossing three different genotypes of mice, a TPO-cre/ERT2; BrafCA/wt; Trp53&#916;ex2-10/&#916;ex2-10&#160;transgenic ATC model was developed. The ATC murine model was induced by an intraperitoneal injection of tamoxifen with overexpression of BrafV600E and deletion of Trp53, and the tumors were generated within about 1 month. High-resolution ultrasound was applied to investigate the tumor initiation and progression, and the dynamic growth curve was obtained by measuring the tumor sizes. Compared to magnetic resonance imaging (MRI) and computed tomography scanning, ultrasound has advantages in observing the ATC murine model, such as being noninvasive, portable, in real-time, and without radiation exposure. High-resolution ultrasound is suitable for dynamic and multiple measurements. However, ultrasonographic examination of the thyroid in mice requires relevant anatomical knowledge and experience. This article provides a detailed procedure for utilizing high-resolution ultrasound to scan tumors in the transgenic ATC model. Meanwhile, ultrasonic parameter adjustment, ultrasound scanning skills, anesthesia and recovery of the animals, and other elements that need attention during the process are listed.

The present protocol describes high-frequency ultrasonography for visualizing the entire mouse thyroid gland and monitoring the growth of anaplastic thyroid carcinoma.

Anaplastic thyroid carcinoma (ATC) is associated with a poor prognosis and short median survival time, but no effective treatment improves the outcomes ...

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 ...

Standardizing Ovarian Cancer Organoid Biobanking

Strategy for Biobanking of Ovarian Cancer Organoids: Addressing the Interpatient Heterogeneity across Histological Subtypes and Disease Stages

While the establishment of an ovarian cancer biobank from patient-derived organoids along with their clinical background information promises advances in research and patient care, standardization remains a challenge due to the heterogeneity of this lethal malignancy, combined with the inherent complexity of organoid technology. This adaptable protocol provides a systematic framework to realize the full potential of ovarian cancer organoids considering a patient-specific variability of progenitors. By implementing a structured experimental workflow to select optimal culture conditions and seeding methods, with parallel testing of direct 3D seeding versus a 2D/3D route, we obtain, in most cases, robust long-term expanding lines suitable for a broad range of downstream applications. 
Notably, the protocol has been tested and proven efficient in a great number of cases (N = 120) of highly heterogeneous starting material, including high-grade and low-grade ovarian cancer and stages of the disease with primary debulking, recurrent disease, and post-neoadjuvant surgical specimens. Within a low Wnt, high BMP exogenous signaling environment, we observed progenitors being differently susceptible to the activation of the Heregulin 1 &#223; (HER&#223;-1)-pathway, with HER&#223;-1 promoting organoid formation in some while inhibiting it in others. For a subset of the patient's samples, optimal organoid formation and long-term growth necessitate the addition of fibroblast growth factor 10 and R-Spondin 1 to the medium. 
Further, we highlight the critical steps of tissue digestion and progenitor isolation and point to examples where brief cultivation in 2D on plastic is beneficial for subsequent organoid formation in the Basement Membrane Extract type 2 matrix. Overall, optimal biobanking requires systematic testing of all main conditions in parallel to identify an adequate growth environment for individual lines. The protocol also describes the handling procedure for efficient embedding, sectioning, and staining to obtain high-resolution images of organoids, which is required for comprehensive phenotyping.

Author Spotlight: Advancing Personalized Medicine in Ovarian Cancer 

This protocol offers a systematic framework for the establishment of ovarian cancer organoids from different disease stages and addresses the challenges of patient-specific variability to increase yield and enable robust long-term expansion for subsequent applications. It includes detailed steps for tissue processing, seeding, adjusting media requirements, and immunofluorescence staining.

While the establishment of an ovarian cancer biobank from patient-derived organoids along with their clinical background information promises advances in ...

Real-Time Tracking of Nanoparticles in Brain Tumors Using 2P-IVM in Mice

Two-Photon Intravital Microscopy of Glioblastoma in a Murine Model

The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the accumulation and distribution of macromolecules in brain tumors in vivo would greatly enhance our ability to understand and optimize drug delivery in preclinical models. This protocol describes a method for real-time in vivo tracking of intravenously administered fluorescent-labeled nanoparticles with two-photon intravital microscopy (2P-IVM) in a mouse model of glioblastoma (GBM).
The protocol contains a multi-step description of the procedure, including anesthesia and analgesia of experimental animals, creating a cranial window, GBM cell implantation, placing a head bar, conducting 2P-IVM studies, and post-surgical care for long-term follow-up studies. We show representative 2P-IVM imaging sessions and image analysis, examine the advantages and disadvantages of this technology, and discuss potential applications.
This method can be easily modified and adapted for different research questions in the field of in vivo preclinical brain imaging.

Author Spotlight: Multimodal Imaging Strategies for Optimizing Drug Delivery and Early Detection in Glioblastoma Treatment

We present a novel approach for two-photon microscopy of the tumor delivery of fluorescent-labeled iron oxide nanoparticles to glioblastoma in a mouse model.

The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the ...