Name: longitudinal intravital imaging through clear silicone windows
Uploaded: 2022-01-05T12:30:00
Description: Watch this Scientific Journal Video about Longitudinal Intravital Imaging Through Clear Silicone Windows at JoVE.com

We thank Rob Eifert for his assistance in designing and optimizing the laser-cut stainless steel grids. This work was supported by the CSHL Cancer Center (P30-CA045508) and funds for M.E. from the National Institutes of Health (NIH) (1R01CA2374135 and 5P01CA013106-49); CSHL and Northwell Health; the Thompson Family Foundation; Swim Across America; and a grant from the Simons Foundation to CSHL. M.S. was supported by the National Institute of General Medical Sciences Medical Scientist Training Program Training Award (T32-GM008444) and the National Cancer Institute of the NIH under award number 1F30CA253993-01. L.M. is supported by a James S. McDonnell Foundation Postdoctoral Fellowship. J.M.A. is the recipient of a Cancer Research Institute/Irvington Postdoctoral Fellowship (CRI Award #3435). D.A.T. is supported by the Lustgarten Foundation Dedicated Laboratory for Pancreatic Cancer Research and the Thompson Family Foundation. Cartoons were created with Biorender.com.

All procedures described were performed in accordance with the Cold Spring Harbor Laboratory Surgical Guidelines and had been approved by the Institutional Animal Care and Use Committee at Cold Spring Harbor Laboratory.
1. Casting the silicone window 
<ol>
	<li>Prepare the silicone polymer (PDMS) by mixing the base elastomer and curing agent in a 10:1 (v/v) ratio.</li>
	<li>Cast a window by depositing a small quantity of PDMS on a sterile, smooth surface and adjust the volum...

<ol>
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J., Werb, Z., Egeblad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+mice+for+long-term+intravital+imaging+of+the+mammary+gland.">Preparation of mice for long-term intravital imaging of the mammary gland.</a> Cold Spring Harbor Protocols. 2011 (2), 5562 (2011).</li><li>Ritsma, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+implantation+of+an+abdominal+imaging+window+for+intravital+microscopy.">Surgical implantation of an abdominal imaging window for intravital microscopy.</a> Nature Protocols. 8 (3), 583-594 (2013).</li><li>Pittet, M. J., Garris, C. S., Arlauckas, S. 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Intravital imaging windows are important tools for directly visualizing physiological and pathological processes at cellular resolution as they unfold over time. The novel procedure described for casting and inserting flexible, silicone imaging windows in mice overcomes some of the most common issues with currently used imaging windows (exudate, breaking, and interference with normal mobility), provides additional safety for the mouse, and increases the accessibility of this technique.
The mos...

Intravital microscopy (IVM), the imaging of tissues in anesthetized animals, offers insights into the dynamics of physiological and pathological events at cellular resolution in intact tissues. The applications of this technique vary widely, but IVM has been instrumental in the cancer biology field to help elucidate how cancer cells invade tissues and metastasize, interact with the surrounding microenvironment, and respond to drugs1,2,3. In addition, IVM has been key to advancing the understanding of the complex mechanisms governing immune responses by providing insights complementary to ex vivo profiling approaches (e.g., flow cytometry). For instance, intravital imaging experiments have revealed details about immune functions as they relate to cell migration and cell-cell contact&#160;and have offered a platform to quantitate spatiotemporal dynamics in response to injury or infection4,5,6,7. Many of these processes can also be studied through histological staining, but only IVM allows the tracking of dynamic changes. In fact, whereas a histological section offers a snapshot of the tissue at a given time, intravital imaging can track intercellular and subcellular events within the same tissue over time. In particular, progress in fluorescence labeling and the development of molecular reporters have allowed molecular events to be correlated with cellular behaviors, such as proliferation, death, motility, and interaction with other cells or the extracellular matrix. Most IVM techniques are based on fluorescence microscopy, which due to light scattering, makes imaging deeper tissues challenging. The tissue of interest, therefore, often needs to be surgically exposed with an often invasive and terminal procedure. Thus, depending on the organ site, the tissue can be imaged continuously for a period varying from a few to 40 h8. Alternatively, the surgical insertion of a permanent imaging window permits the imaging of the same tissue sequentially over a period of days to weeks7,9.
The development of new imaging windows has been highlighted as a technological need to further improve intravital imaging approaches10. The prototypical intravital imaging window is a metal ring containing a glass coverslip secured to the skin with sutures11. Interference with free movement, the accumulation of exudate, and damage to the glass coverslip are common problems seen with using such windows. Moreover, the prototypical window requires specialized production, and the surgical procedure can require extensive training. To address these issues, polydimethylsiloxane (PDMS), a silicone polymer, which has previously been used in cranial windows for long-term imaging in the brain12, was adapted for use in abdominal organ and mammary gland imaging. Here, a detailed method for generating PDMS-based silicone windows is presented, including how to cast the window around a stainless-steel grid to provide landmarks for repeated imaging of the same tissue regions. Furthermore, a simple, stitch-free surgical procedure for inserting the window over abdominal organs or the mammary gland is described. This new approach overcomes some of the most common issues with currently used imaging windows and increases the accessibility of longitudinal intravital imaging.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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		<tr>
			<td>3M Medipore Soft Cloth Surgical Tape</td>
			<td>3M</td>
			<td>70200770819</td>
			<td></td>
		</tr>
		<tr>
			<td>Silk suture 4-0 PERMA HAND BLACK 1 x 18&#34; RB-2</td>
			<td>Ethicon&#160;</td>
			<td>N267H</td>
			<td></td>
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		<tr>
			<td>ACTB-ECFP mice</td>
			<td>Jackson Laboratory</td>
			<td>22974</td>
			<td></td>
		</tr>
		<tr>
			<td>AEC Substrate Kit, Peroxidase (HRP), (3-amino-9-ethylcarbazole)</td>
			<td>Vector Laboratories&#160;</td>
			<td>SK-4200</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol swabs</td>
			<td>BD&#160;</td>
			<td>326895</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia system</td>
			<td>Molecular Imaging Products Co.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acqknowledge software and sensors&#160;</td>
			<td>BIOPAC</td>
			<td>ACK100W, ACK100M, TSD110</td>
			<td></td>
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		<tr>
			<td>Betadine spray&#160;</td>
			<td>LORIS</td>
			<td>109-08</td>
			<td></td>
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		<tr>
			<td>c-fms-EGFP (MacGreen) mice</td>
			<td>Jackson Laboratory</td>
			<td>18549</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J mice</td>
			<td>Jackson Laboratory</td>
			<td>664</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45 Monoclonal Antibody (30-F11)</td>
			<td>Invitrogen</td>
			<td>14-0451-82</td>
			<td></td>
		</tr>
		<tr>
			<td>CD68 Antibody</td>
			<td>Abcam</td>
			<td>ab125212</td>
			<td></td>
		</tr>
		<tr>
			<td>Curity gauze sponges&#160;</td>
			<td>Covidien</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Anti-Goat IgG H&#38;L (HRP)&#160;</td>
			<td>Abcam</td>
			<td>ab6885</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Anti-Rabbit IgG H&#38;L (HRP)&#160;</td>
			<td>Abcam</td>
			<td>ab97064</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Anti-Rat IgG H&#38;L (HRP)&#160;</td>
			<td>Abcam</td>
			<td>ab102182</td>
			<td></td>
		</tr>
		<tr>
			<td>Dow SYLGARD 184 Silicone Encapsulant Clear</td>
			<td>Electron Microscopy Sciences</td>
			<td>24236-10</td>
			<td>Two-part, 10:1 mixing ratio</td>
		</tr>
		<tr>
			<td>Round Cover Glass, 8mm Diameter, #1.5 Thickness&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>72296-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Ender-3 Pro 3D printer</td>
			<td>Shenzhen Creality 3D Technology Co., LTD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Far Infrared Heated blanket</td>
			<td>Kent Scientific</td>
			<td>RT-0520</td>
			<td></td>
		</tr>
		<tr>
			<td>Fc Receptor Blocker</td>
			<td>Innovex Biosciences</td>
			<td>NB309</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji imaging processing package</td>
			<td></td>
			<td></td>
			<td>https://imagej.net/software/fiji/</td>
		</tr>
		<tr>
			<td>FluoroSpheres carboxylate, 0.04&#181;m, yellow-green (505/515)</td>
			<td>Invitrogen</td>
			<td>F8795</td>
			<td></td>
		</tr>
		<tr>
			<td>Gating system:</td>
			<td>BIOPAC Systems Inc.</td>
			<td></td>
			<td rowspan="7">The components together allow monitoring mouse vitals during imaging and gating image acquisition on mouse respiration. All were acquired from BIOPAC systems.</td>
		</tr>
		<tr>
			<td>Acqknowledge software&#160;</td>
			<td></td>
			<td>ACK100W, ACK100M</td>
		</tr>
		<tr>
			<td>Diff. Amp. Module, C Series&#160;</td>
			<td></td>
			<td>DA100C</td>
		</tr>
		<tr>
			<td>Dual Gating Sys small animal</td>
			<td></td>
			<td>DTU200&#160;</td>
		</tr>
		<tr>
			<td>MP160 for Windows - Analysis system</td>
			<td></td>
			<td>MP160WSW&#160;</td>
		</tr>
		<tr>
			<td>MouseOx Plus 120V&#160;</td>
			<td></td>
			<td>MOX-120V;015000&#160;</td>
		</tr>
		<tr>
			<td>Pressure Pad&#160;</td>
			<td></td>
			<td>TSD110&#160;</td>
		</tr>
		<tr>
			<td>Gelfoam</td>
			<td>Pfizer</td>
			<td>9031508</td>
			<td>Absorbable gelatin sponge</td>
		</tr>
		<tr>
			<td>Hardened fine scissors</td>
			<td>Fine Science Tools</td>
			<td>14090-11</td>
			<td>Two pairs; stainless steel, sharp-sharp 
			tips, straight tip, 26 mm 
			cutting edge, 11 cm length</td>
		</tr>
		<tr>
			<td>Human/Mouse Myeloperoxidase/MPO Antibody</td>
			<td>R&#38;D Systems</td>
			<td>AF3667</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot bead sterilizer</td>
			<td>Fine Science Tools</td>
			<td>18000-45</td>
			<td>Turn on approximately 30 min 
			before use; sterilize tools at &#62;200 
			&#176;C for 30 s</td>
		</tr>
		<tr>
			<td>Imaris&#160;</td>
			<td>Bitplane</td>
			<td>www.bitplane.com</td>
			<td></td>
		</tr>
		<tr>
			<td>Immersion medium Immersol W 2010</td>
			<td>Zeiss</td>
			<td>444969-0000-000&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin Syringes with BD Ultra-Fine needle 6mm x 31G 1 mL/cc</td>
			<td>BD</td>
			<td>324912</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane (Fluriso)</td>
			<td>VetOne</td>
			<td>502017</td>
			<td></td>
		</tr>
		<tr>
			<td>Lycopersicon Esculentum (Tomato) Lectin (LEL, TL), DyLight&#174; 594</td>
			<td>Vector Laboratories&#160;</td>
			<td>DL-1177-1</td>
			<td></td>
		</tr>
		<tr>
			<td>LysM-eGFP mice</td>
			<td>www.mmrrc.org</td>
			<td>012039-MU</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro dissecting forceps</td>
			<td>Roboz</td>
			<td>RS-5135</td>
			<td>Serrated, slight curve, 0.8 mm tip width; 4&#34; length</td>
		</tr>
		<tr>
			<td>Micro dissecting forceps</td>
			<td>Roboz</td>
			<td>RS-5153</td>
			<td>1 x 2 teeth, slight curve, 0.8 mm tip 
			width, 4&#34; length</td>
		</tr>
		<tr>
			<td>MTS MiniBionix II 808</td>
			<td>MTS Systems</td>
			<td></td>
			<td>Servohydraulic material testing machine</td>
		</tr>
		<tr>
			<td>Neutrophil Elastase 680 FAST probe</td>
			<td>PerkinElmer</td>
			<td>NEV11169</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrogen</td>
			<td>General Welding Supply Corp.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen</td>
			<td>General Welding Supply Corp.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polylactic acid filament</td>
			<td>Hatchbox</td>
			<td></td>
			<td>1.75 mm diameter</td>
		</tr>
		<tr>
			<td>ProLong Diamond Antifade Mountant</td>
			<td>Invitrogen</td>
			<td>P36970</td>
			<td></td>
		</tr>
		<tr>
			<td>Puralube ophthalmic ointment</td>
			<td>Dechra&#160;</td>
			<td>NDC17033-211-38</td>
			<td></td>
		</tr>
		<tr>
			<td>Reflex 7 wound clips</td>
			<td>Roboz Surgical</td>
			<td>RS-9255</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel grid</td>
			<td>Fotofab</td>
			<td></td>
			<td>One grid is 0.200 inches in diameter, with a total of 52 individual grid squares that are 0.016 x 0.016 inches. There is 0.003 inches of space between each square.&#160;&#160;</td>
		</tr>
		<tr>
			<td>Surface Treated SterileTissue Culture Plates</td>
			<td>Fisher Scientific</td>
			<td>FB012929</td>
			<td>Lid used as curing surface for imaging windows</td>
		</tr>
		<tr>
			<td>TriM Scope Multiphoton Microscope&#160;</td>
			<td>LaVision BioTec</td>
			<td></td>
			<td>Imaging was done on an upright 2-photon microscope (Trimscope, LaVision BioTec) equipped with two Ti:Sapphire lasers (Mai Tai and InSight, Spectra-Physics) and an optical parametric oscillator. The following Longpass Dichroic Beamsplitters (Chroma) were used to direct the signal towards four photomultipler tubes: 
			T560LP 
			T665LPXXR 
			T495lxpr</td>
		</tr>
		<tr>
			<td>Vetbond</td>
			<td>3M</td>
			<td>70200742529</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR micro cover glass</td>
			<td>VWR</td>
			<td>48404-453</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Intravital imaging through imaging windows can be used to observe, track, and quantify a wide array of cellular and molecular events at single-cell resolution over a period of hours to weeks. Ideal features for an imaging window include: a) limited impact on the well-being of the mouse and the physiology of the tissue; b) durability; c) simplicity of insertion; and d) clear landmarks for repeated imaging of the same region. The result is a versatile, inert silicone window that is easily produced and inserted and that can...

Watch this Scientific Journal Video about Longitudinal Intravital Imaging Through Clear Silicone Windows at JoVE.com

Longitudinal Intravital Imaging Through Clear Silicone Windows

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 ...

This procedure for casting and inserting silicone windows lowers the technical barriers to longitudinal intravital imaging. Our protocol produces sturdy, easily adaptable windows that are well-tolerated by the animals. Using this technique, we have imaged cancer and immune cells in the liver, pancreas, and mammary gland.With minimal adjustments, it could be adapted to many anatomical locations and biological questions. For those trying this technique for the first time, practice releasing small amounts of glue from the syringe. Applying too much glue will cloud the imaging field and make the surgical area difficult to work with.Start casting the silicone window by depositing a small quantity of the freshly prepared silicone polymer, polydimethylsiloxane, of PDMS, on a clean smooth surface, and adjust the volume to area ratio to get the desired thickness. Once the PDMS is deposited on the desired casting surface, lightly press a stainless steel grid into the silicone to provide landmarks for repeated imaging of the same tissue regions. To remove the air bubbles and degas the silicone polymer, place the coated surface in a vacuum desiccator for 45 minutes.When done, cure the polymer in an oven at 75 to 80 degrees Celsius for 45 minutes. Score the polymer at the edges of the mold and use forceps to gently peel the cured windows from the surface used for casting. Place the mouse on the left lateral decubitus position.Prepare the anesthetized mouse for surgery by applying 10%volume by volume povidone iodine solution and 70%volume by volume ethanol three times consecutively on the shaved surgical site. Care must be taken to maintain a sterile surgical field. Next, use sterile scissors and forceps to make a 10 millimeter incision on the right flank at three millimeters below the costal arch.Remove a one square centimeter section of the skin and use a second pair of sterile scissors and forceps to remove a section of the peritoneum slightly smaller than the section of the skin. Deposit a small quantity of surgical adhesive on a sterile well plate and withdraw it with a 31 gauge syringe to apply on the surface of the liver around the edges of the area to be imaged, then position the window on the desired area, and hold it firmly against the liver for approximately two minutes until the adhesive has dried. After folding the edges of the window under the peritoneum deposit a small quantity of the surgical adhesive on the edges of the window that are now under the peritoneum, push down on the peritoneum with forceps to secure it to the window.Similarly, seal the peritoneum by depositing a small quantity of the surgical adhesive before pushing down the skin, then apply glue around the edges of the window to create a rim that will prevent the skin from growing back over the window. To insert the window in the pancreas, place the mouse on the right lateral decubitus position, and make the incision on the left flank before removing a section of the peritoneum as demonstrated earlier. Using sterile cotton swabs soaked with sterile saline, gently pulled the spleen to visualize the pancreas.Reposition the pancreas with the moistened cotton swabs to make more surface area visible through the incision. Once the pancreas is accessed, insert and seal the imaging window on the desired area as explained before. To access the mammary gland, place the mouse in the supine position, and make a 10 millimeter incision medial to one of the inguinal nipples using sterile scissors and forceps.Remove a 0.5 square centimeter section of the skin above the mammary gland, then gently separate the mammary gland from the skin by spreading the sterile scissors between the two surfaces to disrupt adherence. Use a 31 gauge syringe to apply a small quantity of the surgical adhesive on the surface of the mammary gland around the edges of the area to be imaged. Position and hold the window firmly against the mammary gland until the adhesive dries.After folding the edges of the window under the skin, deposit a small quantity of surgical adhesive on the edges of the window that are now under the skin, and push down on the skin with forceps to secure the skin to the window. Apply the glue around the edges of the window to create a rim that will prevent the skin from growing back over the window. Before beginning the imaging session, clean the window from any residual debris by gently wiping it with a cotton swab dipped in 70%ethanol.When utilizing a water immersion lens, apply the ultrasound gel to the window avoiding bubbles. To start the imaging, locate and place the grid in focus through the oculars. Then determine the approximate tissue imaging depth by setting the bottom of the grid to zero.The information will help in locating the corresponding Z-plane in subsequent imaging sessions. To identify the same location over multiple imaging sessions, utilize the squares on the grid as a reference point. During the imaging session, note the grid orientation and the location of each field of view imaged within the grid.The grid can be used to navigate back to specific grid areas and imaging fields in the success of imaging sessions. Following each imaging session remove any residual lens immersion medium and debris by gently wiping the window with a cotton swab dipped in 70%ethanol. The silicone window allows capturing the dynamic immune cell infiltration over time.The representative analysis illustrates the neutrophil infiltration within the remodeling mammary gland tissue at days two and four. The silicone window allows tumors to be visualized at multiple time points over weeks after window insertion. The windows cast with 200 milligrams of PDMS are generally better for surgical implantation as they exhibited significantly greater toughness than the windows cast with 150 milligrams.Of note, the PDMS windows were not as rigid as standard glass, represented by the Young's modulus of the glass windows being significantly greater than PDMS. The window insertion in different organs was well-tolerated as most mice continued to gain weight over time. The most important thing to remember when attempting this procedure is to fully seal the window using glue.This prevents infection, the tissue from drying out, and the displacement of the window. Following the procedure, orthotopic injections can be done through the silicone window, allowing researchers to study a wide variety of topics, such as cell proliferation, cell-cell interactions, and therapeutic efficacy.

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Research

JoVE Journal

Cancer Research

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

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

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

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

Longitudinal Morphological and Physiological Monitoring of Three-dimensional Tumor Spheroids Using Optical Coherence Tomography

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, high-throughput imaging modalities utilizing bright field or fluorescence detection, are unable to resolve the overall 3D structure of the tumor spheroid due to limited light penetration, diffusion of fluorescent dyes and depth-resolvability. Recently, our lab demonstrated the use of optical coherence tomography (OCT), a label-free and non-destructive 3D imaging modality, to perform longitudinal characterization of multicellular tumor spheroids in a 96-well plate. OCT was capable of obtaining 3D morphological and physiological information of tumor spheroids growing up to about 600 &#181;m in height. In this article, we demonstrate a high-throughput OCT (HT-OCT) imaging system that scans the whole multi-well plate and obtains 3D OCT data of tumor spheroids automatically. We describe the details of the HT-OCT system and construction guidelines in the protocol. From the 3D OCT data, one can visualize the overall structure of the spheroid with 3D rendered and orthogonal slices, characterize the longitudinal growth curve of the tumor spheroid based on the morphological information of size and volume, and monitor the growth of the dead-cell regions in the tumor spheroid based on optical intrinsic attenuation contrast. We show that HT-OCT can be used as a high-throughput imaging modality for drug screening as well as characterizing biofabricated samples.

Optical coherence tomography (OCT), a three-dimensional imaging technology, was used to monitor and characterize the growth kinetics of multicellular tumor spheroids. Precise volumetric quantification of tumor spheroids using a voxel counting approach, and label-free dead tissue detection in the spheroids based on intrinsic optical attenuation contrast, were demonstrated.

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, ...

Longitudinal Intravital Imaging of Brain Tumor Cell Behavior in Response to an Invasive Surgical Biopsy

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized treatment determination. However, perturbation of the tumor architecture by biopsy and other invasive procedures has been associated with undesired effects on tumor progression, which need to be studied in depth to further improve the clinical benefit of these procedures. Conventional static approaches, which only provide a snapshot of the tumor, are limited in their ability to reveal the impact of biopsy on tumor cell behavior such as migration, a process closely related to tumor malignancy. In particular, tumor cell migration is the key in highly aggressive brain tumors, where local tumor dissemination makes total tumor resection virtually impossible. The development of multiphoton imaging and chronic imaging windows allows scientists to study this dynamic process in living animals over time. Here, we describe a method for the high-resolution longitudinal imaging of brain tumor cells before and after a biopsy in the same living animal. This approach makes it possible to study the impact of this procedure on tumor cell behavior (migration, invasion, and proliferation). Furthermore, we discuss the advantages and limitations of this technique, as well as the ability of this methodology to study changes in the cancer cell behavior for other surgical interventions, including tumor resection or the implantation of chemotherapy wafers.

Here we describe a method for high-resolution time-lapse multiphoton imaging of brain tumor cells before and after invasive surgical intervention (e.g., biopsy) within the same living animal. This method allows studying the impact of these invasive surgical procedures on tumor cells&#39; migratory, invasive, and proliferative behavior at a single cell level.

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized ...

Assessing Tumor Microenvironment of Metastasis Doorway-Mediated Vascular Permeability Associated with Cancer Cell Dissemination using Intravital Imaging and Fixed Tissue Analysis

The most common cause of cancer related mortality is metastasis, a process that requires dissemination of cancer cells from the primary tumor to secondary sites. Recently, we established that cancer cell dissemination in primary breast cancer and at metastatic sites in the lung occurs only at doorways called Tumor MicroEnvironment of Metastasis (TMEM). TMEM doorway number is prognostic for distant recurrence of metastatic disease in breast cancer patients. TMEM doorways are composed of a cancer cell which over-expresses the actin regulatory protein Mena in direct contact with a perivascular, proangiogenic macrophage which expresses high levels of TIE2 and VEGF, where both of these cells are tightly bound to a blood vessel endothelial cell. Cancer cells can intravasate through TMEM doorways due to transient vascular permeability orchestrated by the joint activity of the TMEM-associated macrophage and the TMEM-associated Mena-expressing cancer cell. In this manuscript, we describe two methods for assessment of TMEM-mediated transient vascular permeability: intravital imaging and fixed tissue immunofluorescence. Although both methods have their advantages and disadvantages, combining the two may provide the most complete analyses of TMEM-mediated vascular permeability as well as microenvironmental prerequisites for TMEM function. Since the metastatic process in breast cancer, and possibly other types of cancer, involves cancer cell dissemination via TMEM doorways, it is essential to employ well established methods for the analysis of the TMEM doorway activity. The two methods described here provide a comprehensive approach to the analysis of TMEM doorway activity, either in na&#239;ve or pharmacologically treated animals, which is of paramount importance for pre-clinical trials of agents that prevent cancer cell dissemination via TMEM.

We describe two methods for assessing transient vascular permeability associated with tumor microenvironment of metastasis (TMEM) doorway function and cancer cell intravasation using intravenous injection of high-molecular weight (155 kDa) dextran in mice. The methods include intravital imaging in live animals and fixed tissue analysis using immunofluorescence.

The most common cause of cancer related mortality is metastasis, a process that requires dissemination of cancer cells from the primary tumor to secondary ...

Comparing Metastatic Clear Cell Renal Cell Carcinoma Model Established in Mouse Kidney and on Chicken Chorioallantoic Membrane

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, one third of ccRCC patients will develop metastases to the lung, which is related to a very poor outcome for patients. Unfortunately, no therapy is available for this deadly stage, because the molecular mechanism of metastasis remains unknown. It has been known for 25 years that the loss of function of the von Hippel-Lindau (VHL) tumor suppressor gene is pathognomonic of ccRCC. However, no clinically relevant transgenic mouse model of ccRCC has been generated. The purpose of this protocol is to introduce and compare two newly established animal models for metastatic ccRCC. The first is renal implantation in the mouse model. In our laboratory, the CRISPR gene editing system was utilized to knock out the VHL gene in several RCC cell lines. Orthotopic implantation of heterogeneous ccRCC populations to the renal capsule created novel ccRCC models that develop robust lung metastases in immunocompetent mice. The second model is the chicken chorioallantoic membrane (CAM) system. In comparison to the mouse model, this model is more time, labor, and cost-efficient. This model also supported robust tumor formation and intravasation. Due to the short 10 day period of tumor growth in CAM, no overt metastasis was observed by immunohistochemistry (IHC) in the collected embryo tissues. However, when tumor growth was extended by two weeks in the hatched chicken, micrometastatic ccRCC lesions were observed by IHC in the lungs. These two novel preclinical models will be useful to further study the molecular mechanism behind metastasis, as well as to establish new, patient-derived xenografts (PDXs) toward the development of novel treatments for metastatic ccRCC.

Metastatic clear cell renal cell carcinoma is a disease without a comprehensive animal model for thorough preclinical investigation. This protocol illustrates two novel animal models for the disease: the orthotopically implanted mouse model and the chicken chorioallantoic membrane model, both of which demonstrate lung metastasis resembling clinical cases.

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, ...

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 ...

Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be involved in differentially regulating cancer metastatic cascade genes and gene products dependent on the cancer type, tissue, and individual patient characteristics. These represent potentially important targets for genetic therapeutics and personalized medicine approaches. The development of rapid screening platforms is essential for the identification of these genetic targets.
The chick chorioallantoic membrane (CAM) is a highly vascularized, collagen rich membrane located under the eggshell that allows for gas exchange in the developing embryo. Due to the location and vascularization of the CAM, we developed it as an intravital human cancer metastasis model that allows for robust human cancer cell xenografting and real-time imaging of cancer cell interactions with the collagen rich matrix and vasculature. 
Using this model, a quantitative screening platform was designed for the identification of novel drivers or suppressors of cancer metastasis. We transduced a pool of head and neck HEp3 cancer cells with a complete human genome shRNA gene library, then injected the cells, at low density, into the CAM vasculature. The cells proliferated and formed single-tumor cell colonies. Individual colonies that were unable to invade into the CAM tissue were visible as a compact colony phenotype and excised for identification of the transduced shRNA present in the cells. Images of individual colonies were evaluated for their invasiveness. Multiple rounds of selections were performed to decreases the rate of false positives. Individual, isolated cancer cell clones or newly engineered clones that express genes of interest were subjected to primary tumor formation assay or cancer cell vasculature co-option analysis. In summary we present a rapid screening platform that allows for anti-metastatic target identification and intravital analysis of a dynamic and complex cascade of events.

This is an effective method to screen for suppressors or drivers of cancer metastasis. Cells, transduced with an expression library, are injected into the chicken chorioallantoic membrane vasculature to form metastatic colonies. Colonies having decreased or increased invasiveness are excised, expanded, reinjected to confirm their phenotype, and finally, analyzed using high throughput sequencing.

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be ...

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor microenvironment is an important step toward understanding mechanisms that regulate tumor progression. While this can be accomplished through current intravital imaging techniques, it remains challenging due to the heterogeneous nature of tissues and the need for spatial context within the experimental observation. To this end, we have developed an intravital imaging workflow that pairs collagen second harmonic generation imaging, endogenous fluorescence from the metabolic co-factor NAD(P)H, and fluorescence lifetime imaging microscopy (FLIM) as a means to non-invasively compartmentalize the tumor microenvironment into basic domains of the tumor nest, the surrounding stroma or ECM, and the vasculature. This non-invasive protocol details the step-by-step process ranging from the acquisition of time-lapse images of mammary tumor models to post-processing analysis and image segmentation. The primary advantage of this workflow is that it exploits metabolic signatures to contextualize the dynamically changing live tumor microenvironment without the use of exogenous fluorescent labels, making it advantageous for human patient-derived xenograft (PDX) models and future clinical use where extrinsic fluorophores are not readily applicable.

The intravital imaging method described here utilizes collagen second harmonic generation and endogenous fluorescence from the metabolic co-factor NAD(P)H to non-invasively segment an unlabeled tumor microenvironment into tumor, stromal, and vascular compartments for in-depth analysis of 4D intravital images.

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor ...

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 ...

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 ...