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. 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The authors have nothing to disclose.

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>
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		<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>
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		<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>
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		<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 ...

This technique can answer key questions in regulatory processes such as which role protein dynamics and organization play in functional adaptation. The main advantage of this technique is that it can be done in living cells, thereby revealing protein dynamics on the single-molecule level. Though this method was originally implemented to provide insights into the spatiotemporal organization of mitochondrial membrane proteins, it can also be applied for other organelles and other membrane proteins.Demonstrating the technique will be Timo Appelhans, a grad student from my lab. After transfecting and preparing cell samples and the microscope according to the text protocol, mount the prepared sample with fluorescent beads in the sample holder between the PTFE ring and the red rubber ring. Start the microscope, all hardware components, and all software needed for microscopy.Use a fresh, lint-free tissue sprayed with isopropanol to clean the objective lens and the bottom of the cover slip. Then place a droplet of immersion oil on the pupil of the objective lens. Next, place the sample holder with the bead sample onto the microscope stage so that the bottom of the cover slip contacts the oil.Then focus on the beads by using transmission light or a laser line. Adjust the power of the two excitation lasers to achieve similar signal density in the two fluorescence channels. Then search for an area with many distinct fluorescent signals.Generate a merged view of the fluorescent channels by using the camera control software. Then use the screws at the image splitter to manually tilt the internal optical image splitter to achieve the best overlay of the signals from the two fluorescent channels. Start the TIRF microscope-controlling software and choose to display individual channels in live stream mode.Take a snapshot image, exciting fluorescents in all channels. Then use this image to produce the transformation matrix. Start the software analysis plugin and load the previously-recorded dual-color images of fluorescent beads into the software.Choose the used orientation of the fluorescent channels. Click Yes when asked for calibrate images, and select the previously-taken snapshot. Open the unit manager to define unit conversion factors, then open the localization manager.To determine the point spread function, or PSF, press the button PSF Radius. In the PSF Estimator window that opens, define the numerical aperture and the emission maximum. Then click Estimate PSF Radius.Next, accept the obtained experimental PSF. Define the evaluation box, number of deflation loops, and how many cores of the computer are used for calculation. Then press Localize to start fitting the intensity distribution of single particles, using a 2D symmetric Gaussian function.Open the calibration manager. In the rendered, merged image of the two channels, the original signal and the localized centers will be shown. Choose Affine mode.Then manually connect the corresponding pairs of localized centers in the two channels, that have originated from the same fluorescent bead, by drawing a connection line. Connect the corresponding signals distributed over the field of view. Then press Accept, and save the transformation matrix.To carry out single molecular imaging of mitochondrial membrane proteins, mount the specimen cover slip between the rubber and PTFE rings. Then fill the chamber with 0.5 to 0.8 milliliters of imaging medium. Clean the objective lens and bottom of the cover slip.Then use transmission light or a laser line to focus on the cells, after adding a drop of oil to the lens. Then adjust the power of the two excitation lasers to achieve a similar intensity, and search for an area with many fluorescent signals. Adjust the illumination angle in the TIRF microscope-controlling software to create an incident angle that is smaller than the critical angle, or the TIR-mode, to excite the specific region of interest via a HILO sheet.Adapting the illumination of the TIRF microscope to excite cell organelles using the HILO illumination requires quick judgment of the signal-to-background ratio and experience in TIRF microscopy, as well as a knowledge of cell morphology. Set the EM gain and choose an exposure time suitable for the experiment that collects sufficient photons per frame. Then adjust the laser power in order to achieve a high signal-to-noise ratio, as this ratio directly corresponds to the localization precision.Find an area in the cell periphery with non-overlapping, elongated mitochondria and single-molecule signals. If no single signals are visible in one or both channels, wait until bleaching results in the appearance of single-molecule signals. In the case only one channel needs to be bleached, switch the laser of the other channel off, to avoid bleaching, before recording this channel.Record until the number of signals is too low for reasonable continuation. Start the imaging-processing software and check for mitochondrial structures by generating a cumulative rendered-sum image of at least 1, 000 recorded frames. Start the software analysis plugin.To carry out data processing for localization and tracking, load the raw data. Check multicolor. Choose the correct orientation of both channels.When asked for Calibrate images, load the transformation matrix. The channels will be separately displayed. Open the raw data from the image container by clicking Show.Then open unit manager in the top tab, and define unit conversion factors in each channel. Open the localization manager in the top tab, and define the evaluation box and number of deflation loops. Click on PSF Radius to open the PSF estimator.Set numerical aperture and emission maximum to get the theoretical PSF value. Press Accept. Repeat this for all channels.Then press Localize to start the single-particle localization for the recorded signals in both channels. Wait for the software to finish. Now, a super-resolved image for each channel is obtained in the image container of the software analysis plugin.PINK1 is an important factor guaranteeing mitochondrial functionality. To determine the localization of PINK1 relative to Tom20 in functional and dysfunctional mitochondria, a time series of 1, 000 frames was recorded. The cumulative image of all signals over time from one channel showed that PINK1 was localized in the cytosol, at the outer membrane, and inside mitochondria under normal conditions, but preferentially in the outer membrane of depolarized mitochondria.As the summed images of localized particles show, PINK1 is distributed mainly inside polarized mitochondria, while Tom20 is localized in the outer mitochondrial membrane. This becomes even clearer in the merged image of localized Tom20 and PINK1 particles, which shows that the distribution of the outer membrane protein Tom20 is much broader. To verify the localization of PINK1 inside mitochondria, the 30 kilodalton subunit of respiratory complex one was fused to photoactivable GFP and recorded by fluorescence photoactivation localization microscopy.The cumulative triple-color image and the cross-section distribution show the overlay of complex one and PINK1, confirming the import of full-length PINK1. These data demonstrate that subpopulations of PINK1 occupy several mitochondrial micro-compartments. Once mastered, this technique can be done in 20 hours, including recording and post-processing of data, if it's done properly.While attempting this procedure, it is important to remember to test for organelle movement. Following this procedure, the relative localization of proteins can be determined. After its development, this technique paved the way for researchers in the field of single-particle tracking of mitochondrial proteins to observe their behavior in Z2, and different metabolic conditions, and under stress.After watching this video, you should have a good understanding of how to perform dual-column microscopy of mitochondrial proteins to reveal their sub-organelle position and trafficking.

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Research

JoVE Journal

Biology

8.4K Views.  University of Osnabrück. This technique can answer key questions in regulatory processes such as which role protein dynamics and organization play in functional adaptation. The main advantage of this technique is that it can be done in living cells, thereby revealing protein dynamics on the single-molecule level. Though this method was originally implemented to provide insights into the spatiotemporal organization of mitochondrial membrane proteins, it can also be applied for other organelles and other membrane prote...