Name: intravital microscopy for imaging subcellular structures in live mice expressing fluorescent proteins
Uploaded: 2013-09-01T19:00:00
Description: Watch this Scientific Journal Video about Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins at JoVE.com

This research was supported by the Intramural Research Program of the NIH, National Institute of Dental and Craniofacial Research.

Part 1: Microscope and Preparation of the Imaging Setup
<ol>
	<li>Any inverted confocal microscope should be suitable for IVM. In this study an Olympus IX81 inverted microscope, equipped with a Fluoview-1000 laser scanning confocal unit was used. To achieve the best possible resolution, the use of high NA objectives, such as the water-immersion UPLSAPO 60x/1.20 NA, and the oil immersion Plan-Apo 60x/1.42 NA is recommended. Oil immersion objectives have higher collection efficiencies and although they require the use of...

<ol>
	<li>Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+in+vivo+analyses+of+cell+function+using+fluorescence+imaging+(*).">Emerging in vivo analyses of cell function using fluorescence imaging (*).</a> Annu Rev Biochem. 80, 327-332 (2011).</li><li>Cukierman, E., Pankov, R., Stevens, D. R., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taking+cell-matrix+adhesions+to+the+third+dimension.">Taking cell-matrix adhesions to the third dimension.</a> Science. 294, 1708-1712 (2001).</li><li>Weigert, R., Sramkova, M., Parente, L., Amornphimoltham, P., Masedunskas, A. <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:+a+novel+tool+to+study+cell+biology+in+living+animals.">Intravital microscopy: a novel tool to study cell biology in living animals.</a> Histochem Cell Biol. 133, 481-491 (2010).</li><li>Dunn, K. W., 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=Functional+studies+of+the+kidney+of+living+animals+using+multicolor+two-photon+microscopy.">Functional studies of the kidney of living animals using multicolor two-photon microscopy.</a> Am. J. Physiol. Cell Physiol. 283, C905-C916 (2002).</li><li>Masedunskas, A., Porat-Shliom, N., Weigert, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulated+exocytosis:+novel+insights+from+intravital+microscopy.">Regulated exocytosis: novel insights from intravital microscopy.</a> Traffic. 13, 627-634 (2012).</li><li>Masedunskas, 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=Role+for+the+actomyosin+complex+in+regulated+exocytosis+revealed+by+intravital+microscopy.">Role for the actomyosin complex in regulated exocytosis revealed by intravital microscopy.</a> Proc. Natl. Acad. Sci. U.S.A. 108, 13552-13557 (2011).</li><li>Masedunskas, A., Weigert, 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+two-photon+microscopy+for+studying+the+uptake+and+trafficking+of+fluorescently+conjugated+molecules+in+live+rodents.">Intravital two-photon microscopy for studying the uptake and trafficking of fluorescently conjugated molecules in live rodents.</a> Traffic. 9, 1801-1810 (2008).</li><li>Sramkova, M., Masedunskas, A., Parente, L., Molinolo, A., Weigert, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+plasmid+DNA+in+the+salivary+gland+epithelium:+novel+approaches+to+study+dynamic+cellular+processes+in+live+animals.">Expression of plasmid DNA in the salivary gland epithelium: novel approaches to study dynamic cellular processes in live animals.</a> Am. J. Physiol. Cell Physiol. 297, C1347-C1357 (2009).</li><li>Sramkova, M., Najman, 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=.">.</a> Current Frontiers and Perspectives in Cell Biology. , (2012).</li><li>Masedunskas, 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=Everything+you+need+to+know+about+intravital+microscopy:+A+practical+guide+on+imaging+intracellular+structures+in+live+animals.">Everything you need to know about intravital microscopy: A practical guide on imaging intracellular structures in live animals.</a> Bioarchitecture. 2, (2012).</li><li>Masedunskas, A., Sramkova, M., Parente, L., Weigert, 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+microscopy+to+image+membrane+trafficking+in+live+rats.">Intravital microscopy to image membrane trafficking in live rats.</a> Methods Mol. Biol. 931, 153-167 (2013).</li><li>Babbey, C. M., Ryan, J. C., Gill, E. M., Ghabril, M. S., Burch, C. R., Paulman, A., Dunn, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+intravital+microscopy+of+hepatic+transport.">Quantitative intravital microscopy of hepatic transport.</a> Intravital. 1, 44-53 (2012).</li><li>Rothstein, E. C., Nauman, M., Chesnick, S., Balaban, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-photon+excitation+microscopy+in+intact+animals.">Multi-photon excitation microscopy in intact animals.</a> J. Microsc. 222, 58-64 (2006).</li><li>Klinger, A., Orzekowsky-Schroeder, R., von Smolinski, D., Blessenohl, M., Schueth, A., Koop, N., Huettmann, G., Gebert, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+morphology+and+functional+dynamics+of+vital+murine+intestinal+mucosa+revealed+by+autofluorescence+2-photon+microscopy.">Complex morphology and functional dynamics of vital murine intestinal mucosa revealed by autofluorescence 2-photon microscopy.</a> Histochem. Cell Biol. 137, 269-278 (2012).</li><li>Peter, B., Van Waarde, M. A., Vissink, A., s-Gravenmade, E. J., Konings, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degranulation+of+rat+salivary+glands+following+treatment+with+receptor-selective+agonists.">Degranulation of rat salivary glands following treatment with receptor-selective agonists.</a> Clin. Exp. Pharmacol. Physiol. 22, 330-336 (1995).</li></ol>

So far subcellular structures have been imaged mostly in in vitro (i.e. cell cultures) or in ex vivo (i.e. organ-cultures, tissue slices, acinar preparations) model systems that often do not recapitulate the characteristics of intact live tissues 6. In this respect, the approach presented here represents a major breakthrough since it enables imaging the dynamics of a specific membrane trafficking step (i.e. regulated exocytosis) in living mice.	
	<p class="jove_...

In the past two decades the advent of live microscopy and the use of fluorescent proteins have led to major breakthroughs on every cellular process imaginable, thus advancing our understanding of cell biology 1. This field has benefited tremendously from the use of mammalian cell cultures that are extremely powerful model systems, particularly when it comes to experimental manipulations. However, they do not often provide a true representation of the biology of complex multicellular organisms 2. This issue has begun to be addressed by the development of intravital microscopy (IVM) that has opened the door to investigating key biological questions in fields such as neurobiology, immunology and tumor biology 3. So far, most of the studies based on IVM have been performed at the levels of tissues and individual cells, without providing any information about the dynamics of subcellular compartments. Recently, our laboratory and others have developed IVM techniques capable of imaging subcellular structures in live rodents 4-7, 13-15 and allowing pharmacological and genetic manipulations in vivo. This approach has been used by us to study membrane trafficking in vivo, and more specifically endocytosis and regulated exocytosis 6,7.
Our experimental model system is based on exposing, stabilizing and imaging the submandibular salivary glands (SGs) of anesthetized rodents. The choice of the SGs as a model organ for IVM is due to the fact that the glands are easily accessible by performing a minor surgery, can be externalized without compromising their physiology, and stabilized to reduce the motion artifacts due to heartbeat and respiration. In addition, SGs can be selectively manipulated genetically by injecting either viral or non-viral based vectors through the salivary duct 8,9. Finally, SGs are exocrine glands composed of polarized epithelial cells, which form the acini and the ducts, myoepithelial cells, and a complex population of stromal cells. For this reason, they are an excellent model to study exocytosis, endocytosis, gene delivery, and actin cytoskeleton, as highlighted in our recent studies 10, and offer the opportunity to study aspects of cell biology such as cell polarity, cell division, cell-cell junctions, and ion channels.
In this paper we describe in detail an imaging protocol for achieving subcellular resolution in the epithelium of the SGs of a live mouse. Specifically, we show how to image the secretory granules in the acinar cells of the SGs during regulated exocytosis. As previously shown, upon stimulation with agonists of the beta-adrenergic receptor, the secretory granules fuse with the apical plasma membrane and gradually collapse, releasing their content into the acinar canaliculi 6. Our goal is to provide the basic tools to investigators with minimal experience in surgical procedures and animal handling, so that they can successfully perform IVM at a subcellular resolution. Since the most challenging part in IVM is the preparation of the animal, here we focus on the description of the basic surgical procedures that are utilized to expose and immobilize the SGs without compromising their function. As for the procedures to label subcellular structures, several strategies, such as systemic delivery of fluorescent probes, use of transgenic animals, or a combination of both, have been described elsewhere 7,11.

<table border="1" fo:keep-together.within-page="always">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagent</td>
		 </tr>
		 <tr>
			<td>Isoflourane (Forane)</td>
			<td>Baxter</td>
			<td>101936-40</td>
			<td>Handle under chemical hood</td>
		 </tr>
		 <tr>
			<td>Ketamine (ketaved)</td>
			<td>Fort Dodge Animal Health</td>
			<td>57457-034-10</td>
			<td>Stock solution 100 mg/ml</td>
		 </tr>
		 <tr>
			<td>Xylazine (Anased)</td>
			<td>Akorn, Decatur</td>
			<td>61311-481-10</td>
			<td>Stock solution 100 mg/ml</td>
		 </tr>
		 <tr>
			<td>Neomycin/Polymyxin B</td>
			<td>Bausch and Lomb</td>
			<td>24208-785-55</td>
			<td>Use to lubricate the eye of the mouse</td>
		 </tr>
		 <tr>
			<td>Carbomer-940</td>
			<td>Ashalnd, Inc.</td>
			<td>4607-1</td>
			<td></td>
		 </tr>
		 <tr>
			<td>D-Sorbitol</td>
			<td>Sigma-Aldrich</td>
			<td>S1876</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Triethanolamine</td>
			<td>Sigma-Aldrich</td>
			<td>90279</td>
			<td>Add drop wise to prevent the solution to solidify</td>
		 </tr>
		 <tr>
			<td>Isoproternol</td>
			<td>Sigma-Aldrich</td>
			<td>16504</td>
			<td>Prepare fresh solutions in saline when needed. Stocks can be stored at -20 &deg;C</td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Instrument</td>
		 </tr>
		 <tr>
			<td>Isoflurane V 1.9 (Vaporizer)</td>
			<td>Braintree Scientific</td>
			<td>190AF</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Portable Downdraft table equipped with HEPA filter</td>
			<td>Hazard Technology</td>
			<td>PDDT</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Heat lamp, Model HL1</td>
			<td>Braintree Scientific</td>
			<td>HL-1 US</td>
			<td></td>
		 </tr>
		 <tr>
			<td>MicroTherma 2T Thermometer</td>
			<td>Braintree Scientific</td>
			<td>TW2</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Operating Scissors (11.5 cm straight</td>
			<td>World Precision Instruments </td>
			<td>5003708-12</td>
			<td></td>
		 </tr>
		 <tr>
			<td>#7 curved tip tweezers</td>
			<td>World Precision Instruments</td>
			<td>14187</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Microscissors</td>
			<td>World Precision Instruments</td>
			<td>503365</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Black Braided Silk suture #4.0</td>
			<td>George &amp; Tiemann &amp; Co</td>
			<td>160-1219-4</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Gauze sponges 2" x 2"</td>
			<td>Tyco Healthcare</td>
			<td>9022</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Lens cleaning tissue</td>
			<td>Olympus</td>
			<td>CL-TISSUE (M97) AX6476</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

intravital microscopy for imaging subcellular structures in live mice expressing fluorescent proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

In the GFP/mTomato mouse, the acini appear as clearly distinct structures, which express cytosolic GFP and membrane-targeted tandem-Tomato peptide (Figure 2, broken line). In individual acini, acinar cells are delineated by the tandem-Tomato peptide. GFP is also detected in the nuclei that are clearly visible inside the acinar cells (Figure 2, arrows). Cytosolic GFP is excluded from the secretory granules that appear as dark circular vesicles of approximately 1-1.5 &mu;m in diameter (<st...

Watch this Scientific Journal Video about Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins at JoVE.com

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Research

JoVE Journal

Biology

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

Fluorescent Labeling of Drosophila Heart Structures

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

Photoconversion of Purified Fluorescent Proteins and Dual-probe Optical Highlighting in Live Cells

Photoconvertible fluorescent proteins (pc-FPs) are a class of fluorescent proteins with "optical highlighter" capability, meaning that the color of ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

Fluorescence Recovery after Photobleaching of Yellow Fluorescent Protein Tagged p62 in Aggresome-like Induced Structures

Fluorescence recovery after photobleaching (FRAP)&#160;is a microscopy technique that can be used to quantify protein mobility in live cells. In a typical ...