Name: stabilized longitudinal in vivo cellular-level visualization of the pancreas in a murine model with a pancreatic intravital imaging window
Uploaded: 2021-05-06T14:30:00
Description: Watch this Scientific Journal Video about Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window at JoVE.com

This study was supported by grant No. 14-2020-002 from the SNUBH Research Fund and by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2020R1F1A1058381, NRF-2020R1A2C3005694).

All procedures described in this paper were conducted in accordance with the 8th edition of the Guide for the Care and Use of Laboratory Animals (2011)26 and approved by the Institutional Animal Care and Use Committee at the Korea Advanced Institute of Science and Technology (KAIST) and Seoul National University Bundang Hospital (SNUBH).
1. Preparation of the window and other materials
<ol>
	<li>Custom design the pancreatic intravital imaging window to secl...

<ol>
	<li>Dimastromatteo, J., Brentnall, T., Kelly, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+in+pancreatic+disease.">Imaging in pancreatic disease.</a> Nature Reviews. Gastroenterology &amp; Hepatology. 14 (2), 97-109 (2017).</li><li>Cote, G. A., Smith, J., Sherman, S., Kelly, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technologies+for+imaging+the+normal+and+diseased+pancreas.">Technologies for imaging the normal and diseased pancreas.</a> Gastroenterology. 144 (6), 1262-1271 (2013).</li><li>Yachida, S., 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=Distant+metastasis+occurs+late+during+the+genetic+evolution+of+pancreatic+cancer.">Distant metastasis occurs late during the genetic evolution of pancreatic cancer.</a> Nature. 467 (7319), 1114-1117 (2010).</li><li>Hardt, P. D., Brendel, M. D., Kloer, H. U., Bretzel, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+pancreatic+diabetes+(type+3c+diabetes)+underdiagnosed+and+misdiagnosed.">Is pancreatic diabetes (type 3c diabetes) underdiagnosed and misdiagnosed.</a> Diabetes Care. 31, 165-169 (2008).</li><li>Baetens, D., 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=Alteration+of+islet+cell+populations+in+spontaneously+diabetic+mice.">Alteration of islet cell populations in spontaneously diabetic mice.</a> Diabetes. 27 (1), 1-7 (1978).</li><li>Holmberg, D., Ahlgren, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+pancreas:+from+ex+vivo+to+non-invasive+technology.">Imaging the pancreas: from ex vivo to non-invasive technology.</a> Diabetologia. 51 (12), 2148-2154 (2008).</li><li>Marciniak, A., 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=Using+pancreas+tissue+slices+for+in+situ+studies+of+islet+of+Langerhans+and+acinar+cell+biology.">Using pancreas tissue slices for in situ studies of islet of Langerhans and acinar cell biology.</a> Nature Protocols. 9 (12), 2809-2822 (2014).</li><li>Ravier, M. A., Rutter, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+mouse+pancreatic+islets+for+ex+vivo+imaging+studies+with+trappable+or+recombinant+fluorescent+probes.">Isolation and culture of mouse pancreatic islets for ex vivo imaging studies with trappable or recombinant fluorescent probes.</a> Methods in Molecular Biology. 633, 171-184 (2010).</li><li>Frikke-Schmidt, H., Arvan, P., Seeley, R. J., Cras-Meneur, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+in+vivo+imaging+method+for+individual+islets+across+the+mouse+pancreas+reveals+a+heterogeneous+insulin+secretion+response+to+glucose.">Improved in vivo imaging method for individual islets across the mouse pancreas reveals a heterogeneous insulin secretion response to glucose.</a> Science Reports. 11 (1), 603 (2021).</li><li>Lee, E. M., 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=Effect+of+resveratrol+treatment+on+graft+revascularization+after+islet+transplantation+in+streptozotocin-induced+diabetic+mice.">Effect of resveratrol treatment on graft revascularization after islet transplantation in streptozotocin-induced diabetic mice.</a> Islets. 10 (1), 25-39 (2018).</li><li>Evgenov, N. V., Medarova, Z., Dai, G., Bonner-Weir, S., Moore, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+islet+transplantation.">In vivo imaging of islet transplantation.</a> Nature Medicine. 12 (1), 144-148 (2006).</li><li>Mojibian, M., 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=Implanted+islets+in+the+anterior+chamber+of+the+eye+are+prone+to+autoimmune+attack+in+a+mouse+model+of+diabetes.">Implanted islets in the anterior chamber of the eye are prone to autoimmune attack in a mouse model of diabetes.</a> Diabetologia. 56 (10), 2213-2221 (2013).</li><li>Pittet, M. J., Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging.">Intravital imaging.</a> Cell. 147 (5), 983-991 (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>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=Intravital+microscopy+through+an+abdominal+imaging+window+reveals+a+pre-micrometastasis+stage+during+liver+metastasis.">Intravital microscopy through an abdominal imaging window reveals a pre-micrometastasis stage during liver metastasis.</a> Science Translational Medicine. 4 (158), (2012).</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=Intestinal+crypt+homeostasis+revealed+at+single-stem-cell+level+by+in+vivo+live+imaging.">Intestinal crypt homeostasis revealed at single-stem-cell level by in vivo live imaging.</a> Nature. 507 (7492), 362-365 (2014).</li><li>Dolensek, J., Rupnik, M. S., Stozer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+similarities+and+differences+between+the+human+and+the+mouse+pancreas.">Structural similarities and differences between the human and the mouse pancreas.</a> Islets. 7 (1), 1024405 (2015).</li><li>Park, I., Hong, S., Hwang, Y., Kim, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+pancreatic+imaging+window+for+stabilized+longitudinal+in+vivo+observation+of+pancreatic+islets+in+murine+model.">A Novel pancreatic imaging window for stabilized longitudinal in vivo observation of pancreatic islets in murine model.</a> Diabetes &amp; Metabolism Journal. 44 (1), 193-198 (2020).</li><li>Park, I., 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=Neutrophils+disturb+pulmonary+microcirculation+in+sepsis-induced+acute+lung+injury.">Neutrophils disturb pulmonary microcirculation in sepsis-induced acute lung injury.</a> The European Respiratory Journal. 53 (3), 1800786 (2019).</li><li>Park, I., 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=Intravital+imaging+of+a+pulmonary+endothelial+surface+layer+in+a+murine+sepsis+model.">Intravital imaging of a pulmonary endothelial surface layer in a murine sepsis model.</a> Biomedical Optics Express. 9 (5), 2383-2393 (2018).</li><li>Seo, H., Hwang, Y., Choe, K., Kim, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+quantitation+of+injected+circulating+tumor+cells+from+great+saphenous+vein+based+on+video-rate+confocal+microscopy.">In vivo quantitation of injected circulating tumor cells from great saphenous vein based on video-rate confocal microscopy.</a> Biomedical Optics Express. 6 (6), 2158-2167 (2015).</li><li>Moon, J., 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=Intravital+longitudinal+imaging+of+hepatic+lipid+droplet+accumulation+in+a+murine+model+for+nonalcoholic+fatty+liver+disease.">Intravital longitudinal imaging of hepatic lipid droplet accumulation in a murine model for nonalcoholic fatty liver disease.</a> Biomedical Optics Express. 11 (9), 5132-5146 (2020).</li><li>Hwang, Y., 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=In+vivo+cellular-level+real-time+pharmacokinetic+imaging+of+free-form+and+liposomal+indocyanine+green+in+liver.">In vivo cellular-level real-time pharmacokinetic imaging of free-form and liposomal indocyanine green in liver.</a> Biomedical Optics Express. 8 (10), 4706-4716 (2017).</li><li>Hara, M., 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=Transgenic+mice+with+green+fluorescent+protein-labeled+pancreatic+beta+-cells.">Transgenic mice with green fluorescent protein-labeled pancreatic beta -cells.</a> American Journal of Physiology, Endocrinology and Metabolism. 284 (1), 177-183 (2003).</li><li>Lieber, M., Mazzetta, J., Nelson-Rees, W., Kaplan, M., Todaro, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+continuous+tumor-cell+line+(panc-1)+from+a+human+carcinoma+of+the+exocrine+pancreas.">Establishment of a continuous tumor-cell line (panc-1) from a human carcinoma of the exocrine pancreas.</a> International Journal of Cancer. 15 (5), 741-747 (1975).</li><li>National Institutes of Health. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guide+for+the+Care+and+Use+of+Laboratory+Animals.+Committee+for+the+Update+of+the+Guide+for+the+Care+and+Use+of+Laboratory+Animals.">Guide for the Care and Use of Laboratory Animals. Committee for the Update of the Guide for the Care and Use of Laboratory Animals.</a> The National Academies Collection: Reports funded by National Institutes of Health. , (2011).</li><li>Windelov, J. A., Pedersen, J., Holst, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+anesthesia+dramatically+alters+the+oral+glucose+tolerance+and+insulin+secretion+in+C57Bl/6+mice.">Use of anesthesia dramatically alters the oral glucose tolerance and insulin secretion in C57Bl/6 mice.</a> Physiological Reports. 4 (11), 12824 (2016).</li><li>Kim, M. P., 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=Generation+of+orthotopic+and+heterotopic+human+pancreatic+cancer+xenografts+in+immunodeficient+mice.">Generation of orthotopic and heterotopic human pancreatic cancer xenografts in immunodeficient mice.</a> Nature Protocols. 4 (11), 1670-1680 (2009).</li><li>Cichocki, F., 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=GSK3+inhibition+drives+maturation+of+NK+cells+and+enhances+their+antitumor+activity.">GSK3 inhibition drives maturation of NK cells and enhances their antitumor activity.</a> Cancer Research. 77 (20), 5664-5675 (2017).</li><li>Zhu, S., 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=Monitoring+C-peptide+storage+and+secretion+in+islet+beta-cells+in+vitro+and+in+vivo.">Monitoring C-peptide storage and secretion in islet beta-cells in vitro and in vivo.</a> Diabetes. 65 (3), 699-709 (2016).</li><li>Reissaus, C. A., 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=A+versatile,+portable+intravital+microscopy+platform+for+studying+beta-cell+biology+in+vivo.">A versatile, portable intravital microscopy platform for studying beta-cell biology in vivo.</a> Science Reports. 9 (1), 8449 (2019).</li><li>Kong, K., Guo, M., Liu, Y., Zheng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+animal+models+of+pancreatic+ductal+adenocarcinoma.">Progress in animal models of pancreatic ductal adenocarcinoma.</a> Journal of Cancer. 11 (6), 1555-1567 (2020).</li><li>Bisht, S., Feldmann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+modeling+pancreatic+cancer+and+novel+drug+discovery.">Animal models for modeling pancreatic cancer and novel drug discovery.</a> Expert Opinion in Drug Discovery. 14 (2), 127-142 (2019).</li><li>Herreros-Villanueva, M., Hijona, E., Cosme, A., Bujanda, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+pancreatic+cancer.">Mouse models of pancreatic cancer.</a> World Journal of Gastroenterology. 18 (12), 1286-1294 (2012).</li><li>Feig, C., 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=The+pancreas+cancer+microenvironment.">The pancreas cancer microenvironment.</a> Clinical Cancer Research. 18 (16), 4266-4276 (2012).</li><li>Garcia, P. 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The protocol described here consists of intravital imaging of the pancreas using a novel supporting base integrated pancreatic intravital imaging window modified from an abdominal imaging window. Among the protocols described above, the first critical step is the implantation of the intravital pancreatic imaging window in the mouse. For the application of the glue in the window, it is important to apply the glue between the margin of the window and the cover glass, but not on the pancreatic tissue, as it may significantl...

The pancreas is an abdominal organ with an exocrine function in the digestive tract and an endocrine function of secreting hormones into the bloodstream. High-resolution cellular imaging of the pancreas could reveal the pathophysiology of various diseases involving the pancreas, including pancreatitis, pancreatic cancer, and diabetes mellitus1. Conventional diagnostic imaging tools such as computed tomography, magnetic resolution imaging, and ultrasonography are widely available in the clinical field1,2. However, these imaging modalities are restricted to visualizing only structural or anatomical changes, while alterations at the cellular or molecular level cannot be determined. Given that molecular changes in diabetes mellitus or pancreatic cancer in humans can initiate more than 10 years prior to the diagnosis3,4, the detection of pancreatic diseases from their molecular transition during the latent period has the potential to provide an early diagnosis and a timely intervention. Thus, imaging that will overcome the limitations of resolution and provide valuable insights into the function will remarkably gain attention by providing early diagnosis of pancreatic cancer or advanced identification of the alteration of the islets during the progression of diabetes mellitus5.
In particular with the islets, nuclear imaging, bioluminescence imaging, and optical coherence tomography have been suggested as non-invasive islet imaging techniques6. However, the resolution of these methods is substantially low, with typical values ranging from several tens to hundreds of micrometers, offering a limited capability to detect changes at the cellular level in the islets. On the other hand, previous high-resolution studies of islets were performed under ex vivo7,8 (e.g., slicing or digestion of the pancreas), non-physiologic9 (e.g., exteriorization of the pancreas), and heterotopic conditions10,11,12 (e.g., implantation under the kidney capsule, inside the liver, and in the anterior chamber of the eye), which restricts their interpretation and clinical implications. If in vivo, physiologic, and orthotopic model of high-resolution imaging can be established, it will be a critical platform for the investigation of pancreatic islets.
Intravital imaging, which reveals the pathophysiology at a microscopic resolution level in a live animal, has recently received great attention13. Of the in vivo imaging methods, the development of an abdominal imaging window14, which implants a window into the abdomen of a mouse, has allowed the discovery of novel findings (i.e., a pre-micrometastasis stage of early liver metastasis15 and mechanism of stem cell maintenance in the intestinal epithelium16). Although the abdominal imaging window provides valuable results, the applications of this window for the pancreas and the resulting intravital imaging research based on diseases involving pancreas, have not been extensively investigated.
Unlike the well-defined solid organ characteristics of the human pancreas, the pancreas of a mouse is a diffusely distributed soft tissue-like structure17. Therefore, it is incessantly affected by physiological movements including peristalsis and respiration. A previous study on the application of an abdominal imaging window for the pancreas demonstrated that wandering occurred due to motion-artifacts induced by bowel movements18. Severe blurring was observed in the resulting averaged image, which impeded the visualization and identification of the microscale structures.
Herein, we describe the use of a novel supporting base integrated pancreatic intravital imaging window combined with intravital microscopy19,20 to investigate the longitudinal cellular level events in diseases involving the pancreas. In addition to a detailed description of the methodology in the previous study18, the extended application of pancreatic imaging window for various diseases involving the pancreas will be addressed in this paper. In this protocol, a custom-built video-rate laser-scanning confocal microscopy system was utilized as an intravital microscopy system. Four laser modules (wavelengths at 405, 488, 561, and 640 nm) were utilized as an excitation source, and four channels of emission signals were detected by photomultiplier tubes (PMT) through bandpass filters (BPF1: FF01-442/46; BPF2: FF02-525/50; BPF3: FF01-600/37; BPF4: FF01-685/40). Laser scanning consisted of a rotating polygonal mirror (X-axis) and a galvanometer scanning mirror (Y-axis) that enabled the video-rate scanning (30 frames per second). Detailed information about intravital microscopy has been described in the previous studies10,18,19,20,21,22,23.
In our previous islet study18, we successfully and stably imaged the islets in live mice using a transgenic mouse model (MIP-GFP)24 in which the islets were tagged with GFP. The method enabled high-resolution visualization of the changes in the islets over a period of 1 week. It also facilitated imaging of the same islets for up to 3 weeks, which suggests the feasibility of long-term studies of the pancreatic islets for the functional tracking or monitoring during the pathogenesis of diabetes mellitus18. Furthermore, we developed an orthotopic pancreatic cancer model in which fluorescent pancreatic cancer cells (PANC-1 NucLight Red)25 were directly implanted into the pancreas of the mouse. With the application of the pancreatic intravital imaging window, this model could be utilized as a platform for investigating the cellular and molecular pathophysiology in the tumor microenvironment of pancreatic cancer and for the therapeutic monitoring of novel drug candidates.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor 647 Succinimidyl Esters (NHS esters)</td>
			<td>Invitrogen</td>
			<td>A20006</td>
			<td>Fluorescent probe for conjugate with antibody</td>
		</tr>
		<tr>
			<td>BALB/C Nude</td>
			<td>OrientBio</td>
			<td>BALB/C Nude</td>
			<td>BALB/C Nude</td>
		</tr>
		<tr>
			<td>BD Intramedic polyethylene tubing</td>
			<td>BD Biosciences</td>
			<td>427401</td>
			<td>PE10 catheter for connection with needle</td>
		</tr>
		<tr>
			<td>C57BL/6N</td>
			<td>OrientBio</td>
			<td>C57BL/6N</td>
			<td>C57BL/6N</td>
		</tr>
		<tr>
			<td>Cover glasses circular</td>
			<td>Marienfeld</td>
			<td>0111520</td>
			<td>Cover glass for pancreatic imaging window</td>
		</tr>
		<tr>
			<td>FITC Dextran 2MDa</td>
			<td>Merck (Former Sigma Aldrich)</td>
			<td>FD200S</td>
			<td>For vessel identification</td>
		</tr>
		<tr>
			<td>IMARIS 8.1</td>
			<td>Bitplane</td>
			<td>IMARIS</td>
			<td>Image processing</td>
		</tr>
		<tr>
			<td>Intravital Microscopy</td>
			<td>IVIM tech</td>
			<td>IVM-C</td>
			<td>Intravital Microscopy</td>
		</tr>
		<tr>
			<td>IRIS Scissor</td>
			<td>JEUNGDO BIO &#38; PLANT CO, LTD</td>
			<td>S-1107-10</td>
			<td>This product can be replaced with the product from other company</td>
		</tr>
		<tr>
			<td>Loctite 401</td>
			<td>Henkel</td>
			<td>401</td>
			<td>N-butyl cyanoacrylate glue</td>
		</tr>
		<tr>
			<td>Micro Needle holder</td>
			<td>JEUNGDO BIO &#38; PLANT CO, LTD</td>
			<td>H-1126-10</td>
			<td>This product can be replaced with the product from other company</td>
		</tr>
		<tr>
			<td>Micro rectractor</td>
			<td>JEUNGDO BIO &#38; PLANT CO, LTD</td>
			<td>17004-03</td>
			<td>This product can be replaced with the product from other company</td>
		</tr>
		<tr>
			<td>Microforceps</td>
			<td>JEUNGDO BIO &#38; PLANT CO, LTD</td>
			<td>F-1034</td>
			<td>This product can be replaced with the product from other company</td>
		</tr>
		<tr>
			<td>MIP-GFP</td>
			<td>The Jackson Laboratory</td>
			<td>006864</td>
			<td>B6.Cg-Tg(Ins1-EGFP)1Hara/J</td>
		</tr>
		<tr>
			<td>Nylon 4-0</td>
			<td>AILEE</td>
			<td>NB434</td>
			<td>Non-Absorbable Suture</td>
		</tr>
		<tr>
			<td>Omnican N 100 30G</td>
			<td>B BRAUN</td>
			<td>FT9172220S</td>
			<td>For Vascular Catheter, Use only Needle part</td>
		</tr>
		<tr>
			<td>PANC-1 NucLightRed</td>
			<td>Custom-made</td>
			<td>Custom-made</td>
			<td>Made in laboratory</td>
		</tr>
		<tr>
			<td>Pancreatic imaging window</td>
			<td>Geumto Engineering</td>
			<td>Custom order</td>
			<td>Pancreatic imaging window - custom order</td>
		</tr>
		<tr>
			<td>Physiosuite</td>
			<td>Kent Scientific</td>
			<td>PS-02</td>
			<td>Homeothermic temperature controller</td>
		</tr>
		<tr>
			<td>Purified NA/LE Rat Anti-Mouse CD31</td>
			<td>BD Biosciences</td>
			<td>553708</td>
			<td>Antibody for in vivo vessel labeling</td>
		</tr>
		<tr>
			<td>Ring Forceps</td>
			<td>JEUNGDO BIO &#38; PLANT CO, LTD</td>
			<td>F-1090-3</td>
			<td>This product can be replaced with the product from other company</td>
		</tr>
		<tr>
			<td>Rompun</td>
			<td>Bayer</td>
			<td>Rompun</td>
			<td>Anesthetic agent</td>
		</tr>
		<tr>
			<td>TMR Dextran 65-85kDa</td>
			<td>Merck (Former Sigma Aldrich)</td>
			<td>T1162</td>
			<td>For vessel identification</td>
		</tr>
		<tr>
			<td>Window holder</td>
			<td>Geumto Engineering</td>
			<td>Custom order</td>
			<td>Window holder - custom order</td>
		</tr>
		<tr>
			<td>Zoletil</td>
			<td>Virbac</td>
			<td>Zoletil 100</td>
			<td>Anesthetic agent</td>
		</tr>
	</tbody>
</table>

stabilized longitudinal in vivo cellular-level visualization of the pancreas in a murine model with a pancreatic intravital imaging window

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging study, with an abdominal imaging window, enabled visualization of the cellular dynamics in abdominal organs in vivo. However, due to the soft sheet-like architecture of the mouse pancreas that can be easily influenced by physiologic movement (e.g., peristalsis and respiration), it was difficult to perform stabilized longitudinal in vivo imaging over several weeks at the cellular level to identify, track, and quantify islets or cancer cells in the mouse pancreas. Herein, we describe a method for implanting a novel supporting base, an integrated pancreatic intravital imaging window, that can spatially separate the pancreas from the bowel for longitudinal time-lapse intravital imaging of the pancreas microstructure. Longitudinal in vivo imaging with the imaging window enables stable visualization, allowing for the tracking of islets over a period of 3 weeks and high-resolution three-dimensional imaging of the microstructure, as evidenced here in an orthotopic pancreatic cancer model. With our method, further intravital imaging studies can elucidate the pathophysiology of various diseases involving the pancreas at the cellular level.

Intravital microscopy combined with the supporting base integrated pancreatic intravital imaging window enables longitudinal cellular level imaging of the pancreas in a mouse. This protocol with the pancreatic intravital imaging window provides long-term tissue stability that enables the acquisition of high-resolution imaging to track individual islets for up to 3 weeks. As a result, mosaic imaging for an extended field of view, three-dimensional (3D) reconstruction of z-stack imaging, and longitudinal tracking of the sa...

Watch this Scientific Journal Video about Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window at JoVE.com

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

In vivo high-resolution imaging of the pancreas was facilitated with the pancreatic intravital imaging window.

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging ...

This protocol describes stabilized and repetitive cellular-level in vivo imaging of the pancreas with a novel pancreatic intravital imaging window. The main advantage of this technique is that it is possible to perform motionless, three-dimensional imaging with resolution up to the cellular level over three weeks in the pancreas of a live mouse. Begin by preparing the surgical platform and disinfecting the surfaces with 70%ethanol.After anesthetization, use a rectal probe with a homeothermic-controlled heating pad to monitor the body temperature. Remove hair from the left flank of the mouse and apply alcohol and iodine alternatively, rotating from the incision site to the outer surface, never going back to the incision site, and ending with iodine. Then, make a 1.5-centimeter incision on the left flank of the mouse, dissecting the skin and the muscle.Use a black or nylon 4-0 suture to perform a purse string suture in the incision margin. Pull the spleen carefully with ring forceps and identify the pancreas. Placing the window at the flank of the mouse, pass the spleen and the pancreas through the open space of the window.Then, gently place the pancreas on the plate of the imaging window while placing the spleen on the open space of the window. Inject PANC-1 nuclite red directly into the pancreas. Use a 31-gauge catheter needle to apply drops of n-Butyl cyanoacrylate glue to the margin of the imaging window, ensuring a minimal amount of glue is applied.Gently apply a 12-millimeter round cover glass to the margin of the imaging window. Then, hold the suture loop to fit into the lateral groove of the window and tie it three times. Finally, to prevent the interruption of these tight stitches when the mice are awake, cut the maximum proximal site of the tie.Begin by switching on the intravital microscope, including the laser power. To insert a vascular catheter, apply pressure on the proximal side of the tail with the index and third finger. If necessary heat the tail with a lamp.Disinfect the tail vein with a 70%ethanol spray, then insert a 30-gauge catheter into the lateral tail vein and visualize the regurgitation of blood in the PE-10 tube. Apply silk tape on the catheter to stabilize it. Inject FITC dextran and TMR dextran, or other fluorescent probes as appropriate, according to the combination of fluorescent probes.Insert a rectal probe to automatically control the body temperature with a homeothermic heating pad system. Then, insert the pancreatic imaging window prepared during the intravital microscopy setup into the window holder. Transfer the mouse from the surgical platform to the imaging stage.To perform intravital imaging, start with imaging the pancreas at a low magnification, such as four times, to scan the whole view of the pancreas in the pancreatic imaging window. Once the region of interest has been determined, switch to a higher magnification objective lens, like 20 times or 40 times, to perform cellular-level imaging with lateral and axial resolution approximately 0.5 micrometers and 3 micrometers respectively. Perform Z-stack or time-lapse imaging to observe the 3D structure or cellular-level dynamics such as cell migration.Intravital microscopy combined with the pancreatic intravital imaging window provides long-term tissue stability that enables the acquisition of high-resolution imaging to track individual islets for up to three weeks. The window implanted in a C57 black 6 N mouse with intravenously-injected anti-CD31 antibody, conjugated with an Alexa 647 fluorophore, facilitated wide-area imaging and magnified 3D-imaging of the pancreas. Acinar cells were identified in the averaged images of pancreatic tissue in the adjacent vasculature, visualized using autofluorescence and anti-CD31 antibody respectively.Using the mosaic imaging method, which combines a wide-field view with high-resolution imaging, approximately 40 to 50 islets with the adjacent vasculature were visualized in an MIP-GFP mouse. A previous study showed that islets can be tracked for up to three weeks using this stable imaging method. To visualize cancer cells, PANC-1 nuclite red cells were directly implanted into the mouse pancreas during surgery and nearby vessels were stained with anti-CD31 conjugated with Alexa 647.Using this protocol, wide-field images of pancreatic cancer were generated. This helped delineate the margin of the tumor, as well as achieve high-resolution 3D images at the single cell level. The most critical step in this method is the skillful implantation of the pancreatic imaging window in the mouse.Using other combinations of the fluorescent mouse cells, and antibody probes, dynamic interaction of nearby cells with either islets or cancel cells could be identified. This method can be widely applied by those exploring the change of the islet in various pathophysiological conditions and micro-environments of the pancreatic cancer in situ.

Research

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Medicine

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