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.

Research

JoVE Journal

Cancer Research

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