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, ...

intravital-microscopy-for-imaging-subcellular-structures-live-mice

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 developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

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)

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 advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

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

Selection, microinjection, and imaging of fluorescently-labeled F-actin 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

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of &alpha;-actinin in larval hearts. Additionally, we perform labeling of F-actin and &alpha;-actinin in myosin-GFP expressing adult flies and of &alpha;-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.

Fluorescent Labeling of Drosophila Heart Structures

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

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 in live cells. These systems offer a broad selection of fluorescent substrates optimized for a range of imaging instrumentation. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again. There are two steps to using this system: cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. The SNAP-tag is a small protein based on human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein. SNAP-tag labels are dyes conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag. CLIP-tag is a modified version of SNAP-tag, engineered to react with benzylcytosine rather than benzylguanine derivatives. When used in conjunction with SNAP-tag, CLIP-tag enables the orthogonal and complementary labeling of two proteins simultaneously in the same cells.

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again.

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 environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

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 fluorescence can be changed by exposure to light of a specific wavelength. Optical highlighting allows noninvasive marking of a subpopulation of fluorescent molecules, and is therefore ideal for tracking single cells or organelles.
Critical parameters for efficient photoconversion are the intensity and the exposure time of the photoconversion light. If the intensity is too low, photoconversion will be slow or not occur at all. On the other hand, too much intensity or too long exposure can photobleach the protein and thereby reduce the efficiency of photoconversion.
This protocol describes a general approach how to set up a confocal laser scanning microscope for pc-FP photoconversion applications. First, we describe a procedure for preparing purified protein droplet samples. This sample format is very convenient for studying the photophysical behavior of fluorescent proteins under the microscope. Second, we will use the protein droplet sample to show how to configure the microscope for photoconversion. And finally, we will show how to perform optical highlighting in live cells, including dual-probe optical highlighting with mOrange2 and Dronpa.

This protocol describes a general approach to perform photoconversion of fluorescent proteins on a confocal laser scanning microscope. We describe procedures for the photoconversion of puried protein samples, as well as for dual-probe optical highlighting in live cells with mOrange2 and Dronpa.

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

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

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

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

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

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

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+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal 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 FRAP experiment, steady-state fluorescence is observed by repeated imaging with low-intensity laser light. Subsequently, the fluorescent molecules are rapidly and irreversibly impaired via brief exposure to high-intensity laser light. Information about protein mobility is obtained by monitoring the recovery of fluorescence. We used FRAP to determine the mobility of p62 in aggresome-like induced structures (ALIS) in murine macrophages after stimulation with lipopolysaccharide (LPS). Because many existing FRAP protocols are either incomplete or complex, our goal was to provide a comprehensive, practical, and straightforward step-by-step protocol for FRAP experiments with live cells. Here, we describe RAW264.7 macrophage transfection with yellow fluorescent protein-p62 (YFP-p62), induction of ALIS by exposing the cells to LPS, and a step-by-step method for collecting prebleach and postbleach FRAP images and data analysis. Finally, we discuss important factors to consider when conducting a FRAP experiment.

We describe a comprehensive and practical protocol for fluorescence recovery after photobleaching experiments with live cells. Although the protocol was used to measure the mobility of yellow fluorescent protein-tagged p62 in aggresome-like induced structures, it can be applied to a variety of microscopy systems and fluorescent proteins.

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