The authors would like to thank Philip Andresen, Matthew Parent and Sean Carter for computer programming, technical assistance, and useful conversations and Pat Byard for administrative assistance. This work was funded by NIH Career Award K25-AI65459, NIH R15 GM094713, NSF MRI CHE-0722759, Maine Technology Institute MTAF 1106 and 2061, and the Maine Economic Improvement Fund.

Please note: A diagrammatic representation of optical components referenced in this protocol can be found in Figure 1.
1. Cell Sample Preparation
<ol>
	<li>Plate cells at an optimized density (for NIH-3T3 cells, this is roughly 2-5 x 104&#160;cells/cm2) in wells of an 8-well chamber. Cells should be plated in complete media appropriate to the cell type, although media should be made without antibiotics and without phenol red, which contributes to backgr...

<ol>
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Methods. 3, 793-795 (2006).</li><li>Betzig, E., 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=Imaging+intracellular+fluorescent+proteins+at+nanometer+resolution.">Imaging intracellular fluorescent proteins at nanometer resolution.</a> Sci. 313, 1642-1645 (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, 291-308 (2009).</li><li>Gould, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+imaging+of+molecular+positions+and+anisotropies.">Nanoscale imaging of molecular positions and anisotropies.</a> Nat. Methods. 5, 1027-1030 (2008).</li><li>Juette, M. 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Natl. Acad. Sci. U.S.A. 106, 3125-3130 (2009).</li><li>Huang, B., Wang, W. Q., Bates, M., Zhuang, X. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+super-resolution+imaging+by+stochastic+Opt.+reconstruction+microscopy.">Three-dimensional super-resolution imaging by stochastic Opt. reconstruction microscopy.</a> Sci. 319, 810-813 (2008).</li><li>Hess, S. 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=Dynamic+clustered+distribution+of+hemagglutinin+resolved+at+40+nm+in+living+cell+membranes+discriminates+between+raft+theories.">Dynamic clustered distribution of hemagglutinin resolved at 40 nm in living cell membranes discriminates between raft theories.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 17370-17375 (2007).</li><li>Manley, 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=High-density+mapping+of+single-molecule+trajectories+with+photoactivated+localization+microscopy.">High-density mapping of single-molecule trajectories with photoactivated localization microscopy.</a> Nat. Methods. 5, 155-157 (2008).</li><li>Shroff, H., Galbraith, C. G., Galbraith, J. A., Betzig, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+photoactivated+localization+microscopy+of+nanoscale+adhesion+dynamics.">Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics.</a> Nat. 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U.S.A. 104, 20308-20313 (2007).</li><li>Bock, H., 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+far-field+fluorescence+nanoscopy+based+on+photoswitchable+emitters.">Two-color far-field fluorescence nanoscopy based on photoswitchable emitters.</a> Appl. Phys. B. 88, 161-165 (2007).</li><li>Bossi, 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=Multicolor+far-field+fluorescence+nanoscopy+through+isolated+detection+of+distinct+molecular+species.">Multicolor far-field fluorescence nanoscopy through isolated detection of distinct molecular species.</a> Nano Lett. 8, 2463-2468 (2008).</li><li>Gunewardene, M. 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V., 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=Actin+mediates+the+nanoscale+membrane+organization+of+the+clustered+membrane+protein+influenza+hemagglutinin.">Actin mediates the nanoscale membrane organization of the clustered membrane protein influenza hemagglutinin.</a> Biophys. J. , (2013).</li><li>Tanaka, K. 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=Membrane+molecules+mobile+even+after+chemical+fixation.">Membrane molecules mobile even after chemical fixation.</a> Nat. Methods. 7, 865-866 (2010).</li><li>Beisker, W., Dolbeare, F., Gray, J. 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S.T.H. and M.J.M. hold patents in super-resolution microscopy. S.T.H. serves on the scientific advisory board of Vutara, Inc.

Localization-based super-resolution imaging provides many powerful capabilities for biological imaging. The route from individual optical components placed on the table to a functional super-resolution microscope capable of simultaneously imaging multiple fluorescent species in a biological sample presents a number of challenges. Some aspects of the alignment are more critical than others; we endeavor below to provide guidance to prospective users dealing with the most difficult aspects of the route.
<p class="jove_s...

While cellular structures exist on a wide range of spatial scales, fluorescence imaging of cellular organization on length scales smaller than ~250 nm is restricted in conventional microscopy due to the physical constraint of the diffraction limit. This limit was overcome with the advent of fluorescence photoactivation localization microscopy (FPALM1) and similar techniques2,3,&#160;which can localize large numbers of individual molecules with precision of ~10 nm, to generate images with resolution of a few tens of nanometers. FPALM is based on using optical control to activate and inactivate subsets of molecules (for a full description of FPALM, and instructions on how to implement this imaging system, see Gould et al.4). This technique allows for the spatial distributions of whole populations of single molecules to be mapped, thereby elucidating biological structures across length scales spanning from tens of nanometers to tens of microns. Localization-based super-resolution microscopy (hereto referred to as localization microscopy) has now been adapted to address a range of biological questions, with technological developments permitting, for example, the imaging of individual molecular orientations with polarization FPALM, or P-FPALM5,&#160;the fluorescence imaging of single molecules in three dimensions with Biplane FPALM6&#160;or other techniques7-9,&#160;and the super-resolution fluorescence imaging of single molecules in living cells10-12.&#160;Localization microscopy has also been applied to the imaging of multiple species in fixed cells13-16.&#160;Recently, three protein species have been simultaneously imaged with FPALM in both fixed and living cells17.&#160;Localization microscopy can image samples labeled in a variety of ways: examples include proteins expressed with PAFP or PSFP fusion tags, antibodies or molecules labeled with caged organic dyes, or conventional organic dyes. While the use of conventional fluorescent dyes allows for the labeling of proteins in the absence of a fusion-protein tag, the conditions generally required for the use of noncaged organic dyes in super-resolution imaging require samples to be immersed in reducing buffers2.&#160;Additionally, the intracellular delivery of antibody-dye conjugates typically requires cells to be fixed and their membranes permeabilized, or requires that living cells are made permeable through electroporation or some other means. The requirements for reducing buffer conditions and membrane permeabilization limit the suitability of organic dyes for live cell imaging, although recent developments have allowed for effective use of HaloTags and FPALM to image membrane structures18.
FPALM was the first localization microscopy technique to be applied to live cells10.&#160;In live cells, in addition to providing a time dependent spatial map of the locations of labeled molecules, FPALM can track single molecules over multiple frames, and molecular trajectories determined over timescales of milliseconds19.&#160;Thus, FPALM provides access to fairly short timescales and nanoscale resolution.
Multicolor FPALM can be used for a variety of different probes, including photoactivatable proteins and organic caged or noncaged dyes. We here provide detail on the protocol and setup for the simultaneous imaging of two fluorescent protein species, Dendra2 and PAmCherry. We report the outcomes of imaging PAmCherry conjugated to beta actin (PAmCherry-actin) and Dendra2 conjugated to influenza hemagglutinin (Dendra2-HA) in NIH-3T3 fibroblasts. Components described in the setup can be interchanged for other hardware more suited to the imaging of other probes. Where this is the case, we have tried to be explicit in the text.
Multicolor FPALM is ideal for reporting the spatial distributions of multiple protein species in living or fixed cells. This technique is especially suited to investigating spatial and/or dynamic relationships on nanometer length spatial scales, although images will report localization on a range of length scales, from tens of nanometers up to tens of microns. One major advantage of multicolor FPALM is that the setup is relatively inexpensive to construct, and very flexible for use with various probe combinations. The process of construction and calibration of the system from components also provides considerable understanding of factors which can compromise the quality and interpretability of the data, and so the research outcome. We here detail the methods for the optical setup, sample preparation, and data acquisition of multiple protein species, with PSFP and PAFP fusion constructs, using FPALM. While this protocol describes the analysis of fixed cells, these procedures are readily applicable to the imaging of living cells.
The optical setup here described is ideal for the simultaneous imaging of the PSFP Dendra2 and the PAFP PAmCherry. Many other probes may be used for multicolor imaging; however, the precise components required may vary, depending on the excitation and emission spectra of the chosen probes. Choices of dichroic mirrors, filters, and laser wavelengths should be made based on these considerations.

<table border="0" cellpadding="0" cellspacing="0" width="615">
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">LabTek II chambers</td>
			<td style="width:101px;">Nunc</td>
			<td style="width:137px;"> </td>
			<td style="width:183px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Fluorescent beads</td>
			<td style="width:101px;">Invitrogen</td>
			<td style="width:137px;">F-8801</td>
			<td style="width:183px;">Beads for calibration</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Tetraspeck beads</td>
			<td style="width:101px;">Invitrogen</td>
			<td style="width:137px;">T-7279</td>
			<td style="width:183px;">Four color beads for calibration</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:195px;">Objective immersion oil</td>
			<td style="width:101px;">Zeiss</td>
			<td style="width:137px;">518F</td>
			<td style="width:183px;">Immersion oil for high NA objective (dependent on choice of objective)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">HPLC water</td>
			<td style="width:101px;">Fisher Scientific</td>
			<td style="width:137px;">W5-4</td>
			<td style="width:183px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Media</td>
			<td style="width:101px;">ATCC</td>
			<td style="width:137px;">30-2003</td>
			<td style="width:183px;">Or Cellgro 10-090</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Antibiotics</td>
			<td style="width:101px;">GIBCO</td>
			<td style="width:137px;">15070-063</td>
			<td style="width:183px;"> </td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">serum</td>
			<td style="width:101px;">Thermo Scientific</td>
			<td style="width:137px;">SH30087.03</td>
			<td style="width:183px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Lipofectamine</td>
			<td style="width:101px;">Invitrogen</td>
			<td align="right" style="width:137px;">52887</td>
			<td style="width:183px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Optimem I</td>
			<td style="width:101px;">GIBCO</td>
			<td style="width:137px;">11058-021</td>
			<td style="width:183px;"> </td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Trypsin</td>
			<td style="width:101px;">MPBiomedicals</td>
			<td align="right" style="width:137px;">1689149</td>
			<td style="width:183px;"> </td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">paraformaldehyde</td>
			<td style="width:101px;">Fisher Scientific</td>
			<td style="width:137px;">AA433689M</td>
			<td style="width:183px;">CAUTION: Toxic</td>
		</tr>
	</tbody>
</table>

simultaneous multicolor imaging of biological structures with fluorescence photoactivation localization microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

Influenza hemagglutinin (HA) forms clusters on the order of tens of nanometers to micrometers, and these clusters variably colocalize with actin (Figure 5). These spatial distributions corroborate coarser scale imaging of these two proteins28, and the dependence of the HA spatial distributions on actin19.&#160;Multicolor FPALM images can be further used to describe the density, area and perimeter of these clusters, and the degree of colocalization between the two species at both the...

Watch this Scientific Journal Video about Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy at JoVE.com

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

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8.8K Views.  University of Maine. We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers within single cells.