Name: multi-color localization microscopy of single membrane proteins in organelles of live mammalian cells
Uploaded: 2018-06-30T13:30:00
Description: Watch this Scientific Journal Video about Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells at JoVE.com

The authors would like to thank the Biophysics group and Jacob Piehler at the University of Osnabr&#252;ck for continuous support, Wladislaw Kohl for technical assistance and preparation of material, and the CellNanOs board for providing microscopes for use. The project was funded by the SFB 944.

The following protocol follows the guidelines of the local institution research ethics committee.
1. Methods
<ol>
	<li>Cell culture
	<ol>
		<li>Cultivate cells, for example HeLa cells (human cervix carcinoma), in a T25 cell culture flask containing 5 mL of growth medium at 37 &#176;C and 5% CO2. 
		NOTE: For imaging, split the cells onto prepared coverslips (see steps 1.3 and 1.4) and keep in imaging medium.</li>
	</ol>
	</li>
	<li>Cell transfection</...

<ol>
	<li>Appelhans, T., Richter, C., Wilkens, V., Hess, S., Piehler, J., Busch, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+organization+of+mitochondrial+microcompartments+revealed+by+combining+tracking+and+localization+microscopy.">Nanoscale organization of mitochondrial microcompartments revealed by combining tracking and localization microscopy.</a> Nano Letters. 12 (2), 610-616 (2012).</li><li>Appelhans, T., Busch, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Molecule+Tracking+and+Localization+of+Mitochondrial+Protein+Complexes+in+Live+Cells.">Single Molecule Tracking and Localization of Mitochondrial Protein Complexes in Live Cells.</a> Methods Mol Biol. 1567, 273-291 (2017).</li><li>Rust, M. J., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sub-diffraction-limit+imaging+by+stochastic+optical+reconstruction+microscopy+(STORM).">Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM).</a> Nat Methods. 3 (10), 793-795 (2006).</li><li>Gould, T. J., Verkhusha, V. V., Hess, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+biological+structures+with+fluorescence+photoactivation+localization+microscopy.">Imaging biological structures with fluorescence photoactivation localization microscopy.</a> Nat Protoc. 4 (3), 291-308 (2009).</li><li>Pennacchietti, F., Gould, T. J., Hess, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Probe+Photophysics+in+Localization-Based+Superresolution+Microscopy.">The Role of Probe Photophysics in Localization-Based Superresolution Microscopy.</a> Biophys J. 113 (9), 2037-2054 (2017).</li><li>Wegel, E., G&#246;hler, A., Lagerholm, B. C., Wainman, 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+cellular+structures+in+super-resolution+with+SIM,+STED+and+Localisation+Microscopy:+A+practical+comparison.">Imaging cellular structures in super-resolution with SIM, STED and Localisation Microscopy: A practical comparison.</a> Scientific reports. , (2016).</li><li>Pellett, 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=Two-color+STED+microscopy+in+living+cells.">Two-color STED microscopy in living cells.</a> Biomedical Optics Express. 2 (8), (2011).</li><li>Ishigaki, 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=STED+super-resolution+imaging+of+mitochondria+labeled+with+TMRM+in+living+cells.">STED super-resolution imaging of mitochondria labeled with TMRM in living cells.</a> Mitochondrion. 28, 79 (2016).</li><li>Liss, V., Barlag, B., Nietschke, M., Hensel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-labelling+enzymes+as+universal+tags+for+fluorescence+microscopy,+super-resolution+microscopy+and+electron+microscopy.">Self-labelling enzymes as universal tags for fluorescence microscopy, super-resolution microscopy and electron microscopy.</a> Scientific Reports. 5, 17740 (2015).</li><li>Serg&#233;, A., Bertaux, N., Rigneault, H., Marguet, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+multiple-target+tracing+to+probe+spatiotemporal+cartography+of+cell+membranes.">Dynamic multiple-target tracing to probe spatiotemporal cartography of cell membranes.</a> Nat Methods. 5 (8), (2008).</li><li>Appelhans, T., Busch, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+imaging+of+mitochondrial+membrane+proteins+in+specific+sub-organelle+membrane+locations.">Dynamic imaging of mitochondrial membrane proteins in specific sub-organelle membrane locations.</a> Biophysical reviews. 9 (4), 345-352 (2017).</li><li>Wilmes, 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=Triple-color+super-resolution+imaging+of+live+cells:+resolving+submicroscopic+receptor+organization+in+the+plasma+membrane.">Triple-color super-resolution imaging of live cells: resolving submicroscopic receptor organization in the plasma membrane.</a> Angewandte Chemie. 51 (20), 4868-4871 (2012).</li><li>Niewidok, B., 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=Single-molecule+imaging+reveals+dynamic+biphasic+partition+of+RNA-binding+proteins+in+stress+granules.">Single-molecule imaging reveals dynamic biphasic partition of RNA-binding proteins in stress granules.</a> J Cell Biol. , (2018).</li><li>Wurm, C. A., Jakobs, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+protein+distributions+define+two+sub-compartments+of+the+mitochondrial+inner+membrane+in+yeast.">Differential protein distributions define two sub-compartments of the mitochondrial inner membrane in yeast.</a> FEBS Lett. 580 (24), 5628-5634 (2006).</li><li>Schmidt, R., Wurm, C. A., Punge, A., Egner, A., Jakobs, S., Hell, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+cristae+revealed+with+focused+light.">Mitochondrial cristae revealed with focused light.</a> Nano Lett. 9 (6), 2508-2510 (2009).</li><li>Kukat, C., Wurm, C. A., Spahr, H., Falkenberg, M., Larsson, N. G., Jakobs, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+microscopy+reveals+that+mammalian+mitochondrial+nucleoids+have+a+uniform+size+and+frequently+contain+a+single+copy+of+mtDNA.">Super-resolution microscopy reveals that mammalian mitochondrial nucleoids have a uniform size and frequently contain a single copy of mtDNA.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (33), 13534-13539 (2011).</li><li>Beinlich, F., Drees, C., Piehler, J., Busch, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shuttling+of+PINK1+between+Mitochondrial+Microcompartments+Resolved+by+Triple-Color+Superresolution+Microscopy.">Shuttling of PINK1 between Mitochondrial Microcompartments Resolved by Triple-Color Superresolution Microscopy.</a> ACS chemical biology. 10 (9), 1970-1976 (2015).</li><li>Shim, 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=Super-resolution+fluorescence+imaging+of+organelles+in+live+cells+with+photoswitchable+membrane+probes.">Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes.</a> Proceedings of the National Academy of Sciences. 109 (35), 13978-13983 (2012).</li><li>Sbalzarini, I., Mezzacasa, A., Helenius, A., Koumoutsakos, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Organelle+Shape+on+Fluorescence+Recovery+after+Photobleaching.">Effects of Organelle Shape on Fluorescence Recovery after Photobleaching.</a> Biophysical Journal. 89 (3), 1482-1492 (2005).</li><li>Reits, E., Neefjes, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+fixed+to+FRAP:+measuring+protein+mobility+and+activity+in+living+cells.">From fixed to FRAP: measuring protein mobility and activity in living cells.</a> Nature Cell Biology. 3 (6), E145-E147 (2001).</li><li>Goehring, N., Chowdhury, D., Hyman, A., Grill, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRAP+Analysis+of+Membrane-Associated+Proteins:+Lateral+Diffusion+and+Membrane-Cytoplasmic+Exchange.">FRAP Analysis of Membrane-Associated Proteins: Lateral Diffusion and Membrane-Cytoplasmic Exchange.</a> Biophysical Journal. 99 (8), 2443-2452 (2010).</li><li>Bacia, K., Haustein, E., Schwille, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+correlation+spectroscopy:+principles+and+applications.">Fluorescence correlation spectroscopy: principles and applications.</a> Cold Spring Harbor protocols. 2014 (7), 709-725 (2014).</li><li>Sukhorukov, V., Dikov, D., Busch, K., Strecker, V., Wittig, I., Bereiter-Hahn, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+protein+mobility+in+mitochondrial+membranes+of+living+cells.">Determination of protein mobility in mitochondrial membranes of living cells.</a> Biochimica et biophysica acta. 1798 (11), 2022-2032 (2010).</li><li>Kim, T. K., Eberwine, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+cell+transfection:+the+present+and+the+future.">Mammalian cell transfection: the present and the future.</a> Anal Bioanal Chem. 397 (8), 3173-3178 (2010).</li><li>Graham, F. L., van der Eb, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+technique+for+the+assay+of+infectivity+of+human+adenovirus+5+DNA.">A new technique for the assay of infectivity of human adenovirus 5 DNA.</a> Virology. 52 (2), 456-467 (1973).</li><li>Wedeking, T., 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=Spatiotemporally+Controlled+Reorganization+of+Signaling+Complexes+in+the+Plasma+Membrane+of+Living+Cells.">Spatiotemporally Controlled Reorganization of Signaling Complexes in the Plasma Membrane of Living Cells.</a> Small. 11 (44), 5912-5918 (2015).</li><li>Mironov, S. L., Ivannikov, M. V., Johansson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[Ca2+]i+signaling+between+mitochondria+and+endoplasmic+reticulum+in+neurons+is+regulated+by+microtubules.+From+mitochondrial+permeability+transition+pore+to+Ca2+-induced+Ca2++release.">[Ca2+]i signaling between mitochondria and endoplasmic reticulum in neurons is regulated by microtubules. From mitochondrial permeability transition pore to Ca2+-induced Ca2+ release.</a> J Biol Chem. 280 (1), 715-721 (2005).</li><li>Poot, 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=Analysis+of+mitochondrial+morphology+and+function+with+novel+fixable+fluorescent+stains.">Analysis of mitochondrial morphology and function with novel fixable fluorescent stains.</a> J Histochem Cytochem. 44 (12), 1363-1372 (1996).</li><li>Tokunaga, M., Imamoto, N., Sakata-Sogawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+inclined+thin+illumination+enables+clear+single-molecule+imaging+in+cells+(vol+5,+pg+159).">Highly inclined thin illumination enables clear single-molecule imaging in cells (vol 5, pg 159).</a> Nature Methods. 5 (5), 455 (2008).</li><li>Elmokadem, A., Yu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+Drift+Correction+for+Superresolution+Localization+Microscopy+with+Bayesian+Inference.">Optimal Drift Correction for Superresolution Localization Microscopy with Bayesian Inference.</a> Biophys J. 109 (9), 1772-1780 (2015).</li><li>Barlag, B., 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=Single+molecule+super-resolution+imaging+of+proteins+in+living+Salmonella+enterica+using+self-labelling+enzymes.">Single molecule super-resolution imaging of proteins in living Salmonella enterica using self-labelling enzymes.</a> Sci Rep. 6, 31601 (2016).</li><li>Tokunaga, M., Imamoto, N., Sakata-Sogawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+inclined+thin+illumination+enables+clear+single-molecule+imaging+in+cells.">Highly inclined thin illumination enables clear single-molecule imaging in cells.</a> Nature Methods. 5 (2), 159-161 (2008).</li><li>Mortensen, K. I., Churchman, L. S., Spudich, J. A., Flyvbjerg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+localization+analysis+for+single-molecule+tracking+and+super-resolution+microscopy.">Optimized localization analysis for single-molecule tracking and super-resolution microscopy.</a> Nat Methods. 7 (5), 377-381 (2010).</li><li>Jin, S., Youle, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PINK1-+and+Parkin-Mediated+Mitophagy+at+a+Glance.">PINK1- and Parkin-Mediated Mitophagy at a Glance.</a> Journal of Cell Science. 125, 795-799 (2013).</li><li>Yamano, K., Youle, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PINK1+is+degraded+through+the+N-end+rule+pathway.">PINK1 is degraded through the N-end rule pathway.</a> Autophagy. 9 (11), 1758-1758 (2013).</li><li>Wiedemann, N., 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=Machinery+for+protein+sorting+and+assembly+in+the+mitochondrial+outer+membrane.">Machinery for protein sorting and assembly in the mitochondrial outer membrane.</a> Nature. 424 (6948), 565-571 (2003).</li><li>Thompson, R., Larson, D., Webb, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precise+Nanometer+Localization+Analysis+for+Individual+Fluorescent+Probes.">Precise Nanometer Localization Analysis for Individual Fluorescent Probes.</a> Biophysical Journal. 82 (5), 2775-2783 (2002).</li></ol>

Here, a technique for dual color single molecule localization of mobile membrane proteins was presented. Following the protocol, membrane proteins are fused to self-labeling proteins that react with the rhodamine dyes TMR and SiR conjugated to their respective substrates. Rhodamine dyes are bright and photostable and thus allow for repetitive imaging1. For successful performance, several conditions and critical topics have to be kept in mind.
First, it is important to c...

The goal of this protocol is to provide an imaging method to localize and track single membrane proteins inside live cells. We call this method Tracking and Localization Microscopy (TALM)1,2. Like Stochastic Optical Reconstruction Microscopy (STORM)3 and Fluorescence Photoactivation Localization Microscopy ((F)PALM)4,5, TALM is a single molecule-based fluorescence localization technique. However, it is distinct in the way that the mobility of membrane proteins in combination with repetitive imaging of the same labeled molecule at different positions reveals the microcompartment that is accessible for the mobile protein. In other words, the possible localizations of the protein are set by the architecture of the organelle and by the mobility of the protein1. The method is complementary to various other super-resolution techniques6,7,8 because it reveals localization and trajectory maps by imaging mobile proteins. The labeling is based on using genetically engineered fusion proteins that are per se non-fluorescent. These fusion proteins are self-labeling enzymes that react covalently with a substrate conjugated to a dye. This procedure has the advantage that the labeling degree can be controlled by the amount of added substrate. Furthermore, it allows to vary the color of fluorescence, depending on the chosen conjugated dye. Several self-labeling enzyme-tags are available9. Another advantage of using self-labeling enzyme-tags is, that the conjugated dyes usually are more stable and brighter than fluorescent proteins1 and individual proteins therefore can be recorded longer and more precisely until they are bleached. This allows for the recording of trajectories of mobile proteins and the extraction of diffusion coefficients10,11.
Here, we demonstrate the feasibility of TALM with mitochondrial membrane proteins, but it can also be applied for other intra- and extra-cellular membrane proteins, including different cell types12,13. We show that multi-color TALM further allows for the simultaneous distinction of proteins in different subcompartments in complementation to existing super-resolution fluorescence microscopy techniques14,15,16. TALM is compatible with live cell imaging17. The photo-physics of the chosen rhodamines Tetramethylrhodamine (TMR) and Silicon-Rhodamien (SiR), in particular their brightness and stability, allows to record single membrane proteins over multiple frames providing localization (and trajectory) maps. However, TALM is limited for the localization of soluble proteins with high diffusion coefficients since the motion blur is too high and the collected photons per frame are too low for proper localization. Besides, TALM requires less excitation power than for example STORM or Stimulated Emission Depletion (STED) microscopy6,7, reducing phototoxic effects. This is important, since phototoxic stress often affects organellar morphology18&#160;and thus mobility analysis19. In sum, we present multi-color TALM in living cells as a technique that fills a gap between the localization microscopy methods STORM/STED/(F)PALM and techniques that analyze protein mobility such as fluorescence recovery after photobleaching (FRAP)20,21, fluorescence correlation spectroscopy (FCS)22, and fluorescence cross correlation spectroscopy (FCCS)11,23.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >(2-(4-(2-hydroxyethyl)-1-piperazinyl)-ethanesulfonic acid, 1 M) (HEPES)</td>
			<td >Biochrom</td>
			<td >#1104E</td>
			<td ></td>
		</tr>
		<tr >
			<td >DC1: Dichroid beam splitter&#160;</td>
			<td>Chroma</td>
			<td>640 dcxr</td>
			<td>NC506031</td>
		</tr>
		<tr >
			<td >DC2: Polychroic Mirror, beamsplitter</td>
			<td >Chroma</td>
			<td>zt405/488/561/640rpc</td>
			<td>discontinued</td>
		</tr>
		<tr >
			<td >Dulbecco&#180;s Phosphate-Buffered Saline (PBS) 1x (w/o Ca &#38; Mg)</td>
			<td >Sigma-Aldrich &#38; Co.</td>
			<td >#RNBF8311</td>
			<td></td>
		</tr>
		<tr >
			<td >Earle&#180;s MEM without phenol red, without L-Glutamine and without NaHCO3 containing 1% FBS, 0.1% HEPES, 0.1% NEAA, 0.1% Alanyl-L-Glutamine and 34.78% sodium hydrogen carbonate (NaHCO3 0.75g/l)</td>
			<td ></td>
			<td ></td>
			<td>Imaging medium</td>
		</tr>
		<tr >
			<td >Earle&#180;s minimum essential medium (MEM) with phenol red, containing 1% Fetal Bovine Serum Superior (FBS), 0.1% HEPES (2-(4-(2-hydroxyethyl)-1-piperazinyl)-ethanesulfonic acid, 1 M), and 0.1% non-essential amino acids (NEAA)</td>
			<td ></td>
			<td ></td>
			<td>Growth medium</td>
		</tr>
		<tr >
			<td >EF: Emission filter quadbandpass</td>
			<td>AHF analysentechnik</td>
			<td>F72-866</td>
			<td>Brightline HC 446 nm/523 nm/600 nm/677 nm</td>
		</tr>
		<tr >
			<td >EMCCD camera</td>
			<td >Andor</td>
			<td>Andor iXON 897</td>
			<td>EMCCD camera</td>
		</tr>
		<tr >
			<td >Emission filter QuadView filter cubes, orange</td>
			<td>AHF analysentechnik</td>
			<td>F39-637</td>
			<td>bandpass 582 - 619 nm</td>
		</tr>
		<tr >
			<td >Emission filter QuadView filter cubes, red</td>
			<td>Chroma</td>
			<td></td>
			<td>bandpass 655 - 725 nm (HQ 690/70)</td>
		</tr>
		<tr >
			<td >FBS (Fetal bovine serum) superior</td>
			<td >Biochrom</td>
			<td >S0615</td>
			<td></td>
		</tr>
		<tr >
			<td >Fluorescent beads: TetraSpeck&#8482; Microspheres, 0.1 &#181;m, fluorescent blue/green/orange/dark red</td>
			<td >Thermo Fisher Scientific</td>
			<td >T7279</td>
			<td>fluorescent microspheres</td>
		</tr>
		<tr >
			<td >Glutamine</td>
			<td >Biochrom</td>
			<td >#0951C</td>
			<td></td>
		</tr>
		<tr >
			<td >HeLa cells&#160;</td>
			<td >DSMZ</td>
			<td>ACC-57</td>
			<td>Cervical carcinoma cells from patient Henrietta Lacks</td>
		</tr>
		<tr >
			<td >Hela cells CI::paGFP, stable</td>
			<td >Muster et al., PLOSOne 2010</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Hela cells CV g::Halo7-Tag, stable</td>
			<td >Appelhans et al., NanoLett 2012</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Hela cells Tom20::Halo7-Tag, stable</td>
			<td >Appelhans et al., NanoLett 2012</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Image splitter</td>
			<td >Photometrics</td>
			<td >Dual-View QV2</td>
			<td>image splitter emission</td>
		</tr>
		<tr >
			<td >Imaging processing software&#160;</td>
			<td >ImageJ2 / Fiji</td>
			<td >freeware</td>
			<td></td>
		</tr>
		<tr >
			<td >Immersion Oil - ImmersolTM 518 F (ne = 1.518, ve = 45)</td>
			<td >Carl Zeiss Jena GmbH</td>
			<td >444960-0000-000</td>
			<td></td>
		</tr>
		<tr >
			<td >Inverted epifluorescence microscope</td>
			<td >Olympus IX-71/73/83</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Laser 561 nm, 200 mW</td>
			<td >CrystaLaser</td>
			<td>CL-561-200</td>
			<td>561 nm emission</td>
		</tr>
		<tr >
			<td >Laser 642 nm, 140 mW</td>
			<td >Omicron</td>
			<td >Luxx-642-140</td>
			<td>642 nm emission</td>
		</tr>
		<tr >
			<td >MATLAB</td>
			<td >MathWorks</td>
			<td >version R2013a</td>
			<td></td>
		</tr>
		<tr >
			<td >MEM with Earle&#39;s Balanced Salt Solution 2.2 g/L NaHCO3, stable glutamine w/o PR</td>
			<td >Biochrom</td>
			<td >FG-0385</td>
			<td></td>
		</tr>
		<tr >
			<td >MEM with Earle&#39;s Balanced Salt Solution with 2.2 g/L NaHCO3, stable glutamine, Phenolred</td>
			<td >Biochrom</td>
			<td >FG-0325</td>
			<td></td>
		</tr>
		<tr >
			<td >MitoTracker&#174; Deep Red FM</td>
			<td >Thermo Fisher Scientific</td>
			<td >M22426</td>
			<td>dye</td>
		</tr>
		<tr >
			<td >MitoTracker&#174; Green FM</td>
			<td >Thermo Fisher Scientific</td>
			<td >M7514</td>
			<td>dye</td>
		</tr>
		<tr >
			<td >Multi-mode-optical polarization maintaining monomode fiber</td>
			<td >Pointsource/Qioptiq</td>
			<td></td>
			<td>KineFLEX</td>
		</tr>
		<tr >
			<td >NHS-PEG-MAL, Rapp Polymer&#160;</td>
			<td >Rapp Polymere GmbH T&#252;bingen</td>
			<td ></td>
			<td>coverslip coating</td>
		</tr>
		<tr >
			<td >non-essential amino acids (NEAA)</td>
			<td >Biochrom</td>
			<td >#0802E</td>
			<td></td>
		</tr>
		<tr >
			<td >PEG 800 (Polyethylene glycol) 10 %</td>
			<td >Carl Roth GmbH</td>
			<td >Art No. 0263.1</td>
			<td>coverslip coating</td>
		</tr>
		<tr >
			<td >Penicillin/Streptomycin</td>
			<td >Biochrom</td>
			<td >#0122E</td>
			<td></td>
		</tr>
		<tr >
			<td >Plasmid for PINK1-Halo7-Tag expression</td>
			<td >Beinlich et al., ACS Chemical Biology 2015</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Poly-L-lysine (1.2 mg/ml)</td>
			<td >Sigma-Aldrich &#38; Co.</td>
			<td >Cat. No.P9155</td>
			<td>coverslip coating</td>
		</tr>
		<tr >
			<td >RGD Peptide (Ac-CGRGDS-COOH)</td>
			<td >Coring System Diagnostix GmbH, Gernsheim</td>
			<td ></td>
			<td>coverslip coating / Intergrin receptor motif</td>
		</tr>
		<tr >
			<td >Silicon Rhodamine linked to HaloTag&#174;-Ligand (SiRHTL)&#160;</td>
			<td >personal gift from Kai Johnson</td>
			<td ></td>
			<td>dye</td>
		</tr>
		<tr >
			<td >Software analysis plugin</td>
			<td >self-written C. P. Richter, Biophysik Osnabr&#252;ck</td>
			<td >SLIMFAST 16g</td>
			<td></td>
		</tr>
		<tr >
			<td >Tetramethylrhodamine / SNAP-Cell&#174; TMR-Star linked to SNAP-Ligand (TMRstar)&#160;</td>
			<td >New England Biolab&#174;</td>
			<td >S9105S</td>
			<td>dye</td>
		</tr>
		<tr >
			<td >Tetramethylrhodamine linked to HaloTag&#174;-Ligand (TMRHTL)&#160;</td>
			<td >Promega</td>
			<td >G8251</td>
			<td>dye</td>
		</tr>
		<tr >
			<td >TIRF condensor</td>
			<td >Olympus</td>
			<td>Cell^TIRF MITICO System</td>
			<td>TIRF condensor</td>
		</tr>
		<tr >
			<td >TIRF microscope controlling software</td>
			<td >Olympus cellSens 1.12</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >TIRF objective</td>
			<td>Olympus</td>
			<td>150x oil objective (N.A. 1.45; Olympus UAPO)</td>
			<td></td>
		</tr>
		<tr >
			<td >Trypsin/EDTA 10x</td>
			<td >Biochrom</td>
			<td >#0266</td>
			<td></td>
		</tr>
		<tr >
			<td >Water H2O 99,5 % Rotipuran&#174; Low organic&#160;</td>
			<td >Carl Roth GmbH</td>
			<td >Art. No. HN57.1</td>
			<td></td>
		</tr>
	</tbody>
</table>

multi-color localization microscopy of single membrane proteins in organelles of live mammalian cells

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a super-resolution technique that allows for the determination of the microcompartments that are accessible for proteins by generating localization and tracking maps of these proteins. Moreover, by multi-color localization microscopy, the localization and tracking profiles of proteins in different subcompartments are obtained simultaneously. The technique is specific for live cells and is based on the repetitive imaging of single mobile membrane proteins. Proteins of interest are genetically fused with specific, so-called self-labeling tags. These tags are enzymes that react with a substrate in a covalent manner. Conjugated to these substrates are fluorescent dyes. Reaction of the enzyme-tagged proteins with the fluorescence labeled substrates results in labeled proteins. Here, Tetramethylrhodamine (TMR) and Silicon Rhodamine (SiR) are used as fluorescent dyes attached to the substrates of the enzymes. By using substrate concentrations in the pM to nM range, sub-stoichiometric labeling is achieved that results in distinct signals. These signals are localized with ~15&#8211;27 nm precision. The technique allows for multi-color imaging of single molecules, whereby the number of colors is limited by the available membrane-permeable dyes and the repertoire of self-labeling enzymes. We show the feasibility of the technique by determining the localization of the quality control enzyme (Pten)-induced kinase 1 (PINK1) in different mitochondrial compartments during its processing in relation to other membrane proteins. The test for true physical interactions between differently labeled single proteins by single molecule FRET or co-tracking is restricted, though, because the low labeling degrees decrease the probability for having two adjacent proteins labeled at the same time. While the technique is strong for imaging proteins in membrane compartments, in most cases it is not appropriate to determine the localization of highly mobile soluble proteins.

Multi-color imaging and colocalization analysis can help to determine the sub-organellar localization&#160;of proteins. We demonstrated this earlier with the cytosolic phosphatase and tensin homologue, PINK1, that has different sub-mitochondrial locations due to its processing by mitochondrial proteases17. PINK1 is an important factor guaranteeing mitochondrial functionality34,35. To determine the localizat...

Watch this Scientific Journal Video about Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells at JoVE.com

Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells

Here, we present a protocol for multi-color localization of single membrane proteins in organelles of live cells. To attach fluorophores, self-labeling proteins are used. Proteins, located in different membranes compartments of the same organelle, can be localized with a precision of ~18 nm.

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a ...

Research

JoVE Journal

Biology

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

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 ...

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

Few naturally-occurring plasmids are maintained in mammalian cells.  Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus  (EBV) ...

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, ...

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 ...

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...