We thank E. Arzt for his support through INM, M. Koch for help with the ESEM, and DENS Solutions for providing the microchips. Research in part supported by the Leibniz Competition 2013.

1. Preparation of Labeling and Fixation Reagents
<ol>
	<li>Prepare labeling buffer (BSA-GEL-PBS) using autoclaved phosphate buffered saline, pH 7.4 (PBS). Supplement with 1% BSA (bovine albumin fraction V, biotin-free) and 0.2% gelatin (GEL, from cold water fish skin) and vortex/shake well. 
	Note: Can be stored for ~5 days at 4 &#176;C.</li>
	<li>Prepare washing buffer (BSA-PBS) from autoclaved phosphate buffered saline, pH 7.4. Supplement with 1% BSA and vortex/shake well. Note: Can be stored for ~5 days at 4 &#176;C.</li>
	<li>Prepare 50 mM borate buffer (BB) from 200 mM boric acid stock solution, pH adjusted to 8.3 with 200 mM sodium tetraborate. Note: Can be stored for ~12 months at 4 &#176;C.</li>
	<li>Prepare 0.1 M cacodylate buffer (CB) from 0.2 M sodium cacodylate stock solution, pH adjusted to 7.4, supplemented with 0.1 M sucrose. 
	Note: Opened stock solution can be stored for ~5 days at 4 &#176;C.</li>
	<li>Prepare 0.1 M glycine in PBS (GLY&#8211;PBS). 
	Note: Can be stored for ~2 months at 4 &#176;C.</li>
	<li>Prepare 3% paraformaldehyde in CB (3% PFA) from freshly made or opened vial of 16% PFA, stock solution, EM grade. 
	Note: Opened stock solution can be stored for ~5 days at 4 &#176;C.</li>
	<li>Prepare 2% glutaraldehyde in CB (2% GA) from freshly made (or opened vial of) 25% GA stock solution, EM grade. 
	Note: Opened stock solution can be stored for ~5 days at 4 &#176;C.</li>
	<li>Prepare 300 nM EGF-biotin labeling solution
	<ol>
		<li>Dilute 6 &#181;M EGF-biotin stock solution in BSA-GEL-PBS, use a volume of 100-200 &#181;l per microchip. Use within a few hr.</li>
	</ol>
	</li>
	<li>Prepare 10 nM STR-QD labeling solution
	<ol>
		<li>Centrifuge the streptavidin-QD stock solution for 4 min at 8,000 x g, take supernatant and dilute 1:10 in BB. Different types of QDs can be used, for example, QD655, or QD800. Dilute to final concentration in BSA-GEL-PBS, use 75-100 &#181;l per microchip. Use within a few hr.</li>
	</ol>
	</li>
</ol>
2. Preparation of Silicon Nitride Membrane Microchips
<ol>
	<li>Clean a microscope glass slide, as used for light microscopy, with a clean room tissue and high performance liquid chromatography (HPLC) grade ethanol.</li>
	<li>In a laminar airflow workbench with a low dust level, open a 100 mm diameter Petri dish, and lay the cleaned microscope glass into the dish; leave the dish open.</li>
	<li>With clean tweezers, preferentially coated with carbon fiber or other coating, take 4-10 microchips with thin (50 nm) silicon nitride membranes, one after the other, out of their storage box and onto the glass slide. Grab the microchips gently at their sides and avoid touching the fragile upper side, consisting of the thin silicon nitride membrane. Take great care to always keep the membrane side up and do not touch it. 
	Note: The microchips should be rectangular and have flat edges perpendicular to their silicon nitride covered face, so that they can be gripped easily.</li>
	<li>Close the lid of the petri dish and bring it to a fume hood. Get a new clean room tissue and deposit it beside the petri dish. Prepare two clean 200 ml glass beakers by filling one with 50-70 ml acetone, and the other with a similar volume of ethanol, both of HPLC grade. Transfer the microchips from the glass slide first onto the clean room tissue.</li>
	<li>From the tissue, transfer microchips one by one into the first beaker with acetone for removing the protective resist layer. Wait 2 min, while gently moving the beaker a few times to ensure that the microchips are well rinsed.</li>
	<li>Transfer the microchips directly into the beaker with ethanol, without letting them dry out. Wait 2 min. Then transfer the beaker to the laminar airflow workbench. Get a new clean room tissue.</li>
	<li>Transfer the chips onto the tissue and let them dry for a few min. Transfer the microchips onto the glass slide in the Petri dish, close the dish, and bring it to the plasma cleaner.</li>
	<li>Deposit the glass slide with the microchips into the plasma cleaner. Run a 5 min cleaning program to render the surface of the silicon nitride membrane hydrophilic. 
	Note: Suitable settings applied for our plasma cleaner are: 70 mTorr, gas flow of 11.5 sccm for O2 and 35.0 sccm for Ar, 50 W forward radio frequency (RF) target, 5 W RF range and max. reflected RF.</li>
	<li>Put the glass slide with the microchip back into the petri dish, and close its lid. Take care to keep the microchips sterile from now on.</li>
	<li>Examine the window areas of the microchips under a light microscope for possible membrane ruptures or remaining dirt particles.</li>
	<li>Bring the petri dish to the laminar airflow workbench.</li>
	<li>Place the following items in the laminar airflow workbench: tubes/bottles containing sterilized 0.01% poly-L-lysine solution (PLL, mol. wt 150,000&#8211;300,000, sterile-filtered, and cell culture tested), HPLC grade water, Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, DMEM without serum, a new clean room tissue, a sterile 24-well plate (a 12-well plate can also be used), a sterile 96-well plate, sterile pipet tips, a pipette, and flat, rounded tip, tweezers.</li>
	<li>Take the 24-well plate, and fill one well with PLL, and two wells with HPLC grade water, use an approx. volume of 1 ml per well. Take the 96-well plate and fill for every microchip two wells with serum supplemented DMEM and one well with serum-free DMEM, use an approx. volume of 200 &#181;l per well.</li>
	<li>From now on use tweezers with sterile tips to handle the microchips. Achieve this, for instance, by briefly flaming and subsequently cooling off the tips of the tweezers.</li>
	<li>First transfer the microchips from the glass slide onto the clean room tissue. 
	From the tissue, transfer up to six microchips into the PLL-filled well, incubate the microchips for 5 min. 
	Note: For an experiment with more microchips, fill additional wells in the 24-well plate.</li>
	<li>Subsequently rinse the microchips by placing them, one-by-one, into the first water-filled well, and from there into the second water-filled well. Don&#39;t leave the microchips in the water longer than about a minute to avoid removal of PLL.</li>
	<li>Finally place them individually into the DMEM-filled wells of the 96-well plate. Store the 96-well plate in the CO2 incubator until the cell suspension is prepared.</li>
</ol>
3. Preparation of Cells on Microchips
<ol>
	<li>Perform a subculture of COS7 cells to prepare a cell suspension with a concentration of approx. 5 &#215; 105 cells/ml. Make sure the cells are mono-dispersed.</li>
	<li>Take the 96-well plate with the microchips and add one droplet of cell suspension to each microchip. Wait 5 min.</li>
	<li>Start to examine the window areas of the microchips with an inverted microscope to choose the right moment for transferring it into a new well with serum-free DMEM. 
	Note: A good density of cells is achieved with approximately one cell per 2,500 &#181;m2 area, corresponding to 24 COS7 cells on a window size of 150 x 400 &#181;m. Higher cell densities have the risk to hinder maximal flattening of the cells. Be aware that after a cell has settled down onto the SIN membrane, it needs 3-5 min to develop enough adhesion to resist floating off during transfer into the new well.</li>
	<li>Transfer microchip to the next well when sufficient cells have adhered onto the window. Keep the microchips always with the cells facing up and avoid any touching of this side.</li>
	<li>After the transfer, examine each microchip window again. If too many cells were washed off and the number of remaining cells is insufficient, re-triturate the cell suspension (to break up newly formed cell clumps) and repeat steps 3.1-3.5.</li>
	<li>When all transferred microchips have enough adhering cells on their window areas, transfer the chips into the last well with serum-free DMEM. Place the 96-well plate back into the CO2 incubator and let the cells recover and flatten out for several hr, best O/N.</li>
</ol>
4. EGFR Labeling, Fixation, and Fluorescence Microscopy
<ol>
	<li>For the following steps, use a 96-well and a 24-well plate. Pre-fill wells with the respective solutions, and use one row or column per microchip. The 24- well plate is used under the fume hood for the fixation steps with CB, PFA and GA. Always transfer microchips with the cells facing up and avoid any touching of this side.</li>
	<li>Rinse microchips by placing the microchips in a well with fresh BSA-GEL-PBS, then place 96 well plate in incubator at 37 &#176;C for 5 min.</li>
	<li>Incubate with 300 nM EGF-biotin solution for 3 min at 37 &#176;C. Because the cells will start endocytosis of EGF-labeled EGFRs, proceed as fast as possible to step 4.4.</li>
	<li>Rinse 3 times with PBS, and 1 time with CB (now the 96 well plate is again at RT).</li>
	<li>Incubate in 3% PFA for 10 min.</li>
	<li>Rinse chips 1 time with CB, and 3 times with PBS.</li>
	<li>Incubate in GLY-PBS for 2 min, rinse 2 times with 1 time PBS.</li>
	<li>Incubate in 10 nM STR-QD for 10 min.</li>
	<li>Rinse 4 times with BSA-PBS.</li>
	<li>Submerge a microchip in a glass-bottom 35 mm dish pre-filled with BSA-PBS at RT.</li>
	<li>Acquire differential interference contrast (DIC) images and fluorescence images of QD-labeled cells.
	<ol>
		<li>Use a 40X air objective for overview images of cells labeled with QD655. Use an oil immersion objective (for example, 63X) to allow the most efficient collection of the emitted fluorescence light; this is especially needed when imaging cells labeled with QD800.</li>
		<li>Use a filter cube that exposes the QDs to fluorescence excitation light between 340 and 380 nm, and allows the collection of emitted light above 420 nm suitable for most QDs (Filter cube A in this case).</li>
		<li>Minimize the light intensity to avoid too intense light that might damage the sample. Set the exposure time to at least 300 msec, so that the signals of all QDs are captured, noting that the fluorescence of QDs has a blinking behavior.</li>
		<li>Adjust the light intensity, and the amplification to provide bright images without visible image noise, while minimizing the light intensity. 
		Note: Alternatively, shorter exposure time can be used if image series are taken from the same region. These series can be processed to yield a maximum projection image, leading to averaging and thus noise reduction of the resulting image.</li>
	</ol>
	</li>
	<li>Place the imaged microchip back (cells facing up) into a well of a 96-well plate filled with BSA-PBS.</li>
	<li>Rinse microchips 1 times in CB.</li>
	<li>Incubate in 2% GA for 10 min.</li>
	<li>Rinse 1 time in CB, and 3 times with BSA-PBS.</li>
	<li>Store microchips until ESEM imaging, in BSA-PBS pre-filled wells of a new 96-well plate. For every microchip fill a row of four wells with HPLC-grade water. 
	Note: The samples can be kept at 4 &#176;C for several weeks, however, the best results are achieved when the complete imaging series is performed within two days. QDs will slowly start to detach from the sample, it is therefore not recommended to wait more than 10 days before the ESEM imaging is done. 
	Note: Fluorescence images are stored for future analysis. The positions of the cells in the images can easily be correlated with electron microscopy images via localizing the corners of the SiN window and measuring the positions relative to the corners. 
	Note: It is advisable to print high quality fluorescence images and use them for orientation purposes when recording the ESEM images.</li>
</ol>
5. Wet ESEM-STEM of Whole Cells
<ol>
	<li>Prepare the ESEM for wet STEM
	<ol>
		<li>Mount the gaseous secondary electron detector (GSED), and the Peltier stage, containing the STEM detector (located under the sample).</li>
		<li>Start the flow of cooling water through the stage, and set the temperature to 3 &#176;C.</li>
		<li>Set the stage x-y coordinates to zero, and adjust the working distance to approximately 6 mm.</li>
	</ol>
	</li>
	<li>During the ESEM imaging, keep the 96-well plate with the microchips cool, for instance on frozen cooling elements, in a Styrofoam box.</li>
	<li>For loading a sample into the precooled ESEM-STEM stage, take the 96-well plate out of the cooling container and place it close to the opened ESEM stage. Put a new clean room tissue beside it.</li>
	<li>Quickly rinse a microchip (with the cells facing up) by dipping it four times, each time for about a second, into a well filled with HPLC-grade water.</li>
	<li>Blot the backside of the microchip briefly on the clean room tissue and place the microchip into the precooled stage. Make sure that the cells remain wet, this can be recognized by a reflective surface (drying results in a dull surface).</li>
	<li>Wet the sample surface with 3 &#181;l of cooled HPLC-grade water (make sure not to touch the surface with the pipet tip), and fix the sample in the stage.</li>
	<li>Place three additional 3 &#181;l water droplets on the stage close to the sample.</li>
	<li>Close the specimen chamber</li>
	<li>Purge the specimen chamber by cycling the pressure five times between 800 and 1,500 Pa. End the cycling with 800 Pa.</li>
	<li>Switch the electron beam (30 kV) on and examine the sample under the lowest magnification with both detectors, i.e., the GSED, and the STEM detector to search for the location of the microchip window. 
	Note: If the water layer is too thick, the window may not be visible. In this case, start lowering the pressure to 760 Pa and wait a few min. The window first becomes visible with the GSED.</li>
	<li>Position the SiN membrane window in the center of the image by moving the specimen stage and reduce the pressure to 750-720 Pa. 
	Note: When the water layer is thin enough, the window also becomes visible with the STEM detector.</li>
	<li>From now on use the dark field segment of the STEM detector to record images. First, acquire one or two images showing all cells on the complete window area.</li>
	<li>Compare these ESEM images with the light microscopy images, locate the corners of the SiN window.</li>
	<li>Correlate the coordinate frames of both microscopy modalities by comparing the magnification, rotation, and possible mirroring. Locate the same cells in both images.</li>
	<li>Select very small dirt particles or a cell region with small features to adjust imaging settings, such as the eucentric height, the fine focus and the correction of lens stigmatism. Work at a magnification of at least 50.000X, use an electron probe current around 0.6 nA, a pixel-dwell time of 30 &#181;s, and an image size of 1024 x 884 pixels. 
	Note: Other settings may also be used as long as an increase of the electron dose is avoided. 
	Note: Even when working very clean, the chip surfaces will in practice always contain some dirt particles, because the protocol is not executed in a clean room environment.</li>
	<li>To record STEM images from the QD-labeled cells, refer to the fluorescence images and choose a cell of interest. 
	Note: It is best to start with a cell showing strong QD signals.</li>
	<li>Record a STEM overview image of this cell.</li>
	<li>Go to a peripheral region where the cell border is visible and zoom in to reach 50.000X magnification.</li>
	<li>Record images showing individual QDs, use pixel dwell times of 20-30 &#181;sec. In order to avoid radiation damage, image every region only once with high magnification. 
	Note: Usually, at this stage both, the focus and the correction of the stigmatism, need some fine adjustment.</li>
	<li>After the recording of a sufficient number of high magnification images, zoom-out, and acquire another STEM overview image, depicting the regions just recorded with high-magnification as dark rectangles. 
	Note: These overview images will later serve to correlate ESEM and fluorescence images and to determine the precise location of the high magnification images within the cellular context.</li>
	<li>Proceed to the next cell of interest, start with an overview image, record a series of high magnification images, and end with an overview image.</li>
	<li>At the end of an ESEM imaging session, increase the pressure to 850-900 Pa before starting to vent the chamber. 
	Note: This increase of pressure improves the chance of recovering the microchip with a still wetted surface; however, this cannot always be achieved. A sample that has never been dried out can be restored in BSA-PBS and, if desired, reinvestigated in the ESEM at a later point in time.</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+protein+FRET:+the+good,+the+bad+and+the+ugly.">Fluorescent protein FRET: the good, the bad and the ugly.</a> Trends Biochem. Sci. 32, 407-414 (2007).</li><li>Warren, C. M., Landgraf, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+through+ERBB+receptors:+Multiple+layers+of+diversity+and+control.">Signaling through ERBB receptors: Multiple layers of diversity and control.</a> Cell. Signal. 18, 923-933 (2006).</li><li>Leuchowius, K. 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=Parallel+visualization+of+multiple+protein+complexes+in+individual+cells+in+tumor+tissue.">Parallel visualization of multiple protein complexes in individual cells in tumor tissue.</a> Mol. Cell. Proteomics. 12, 1563-1571 (2013).</li><li>Hoenger, A., McIntosh, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+macromolecular+organization+of+cells+by+electron+tomography.">Probing the macromolecular organization of cells by electron tomography.</a> Curr. Opin. Cell Biol. 21, 89-96 (2009).</li><li>Zhang, J., Leiderman, K., Pfeiffer, J. R., Wilson, B. S., Oliver, J. M., Steinberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+the+topography+of+membrane+receptors+and+signaling+molecules+from+spatial+patterns+obtained+using+nanometer-scale+electron-dense+probes+and+electron+microscopy.">Characterizing the topography of membrane receptors and signaling molecules from spatial patterns obtained using nanometer-scale electron-dense probes and electron microscopy.</a> Micron. 37, 14-34 (2006).</li><li>Pinto da Silva, P., Kan, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-fracture:+a+method+for+high+resolution+labeling+of+cell+surfaces.">Label-fracture: a method for high resolution labeling of cell surfaces.</a> J. Cell Biol. 99, 1156-1161 (1984).</li><li>Bergersen, L. H., Storm-Mathisen, J., Gundersen, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunogold+quantification+of+amino+acids+and+proteins+in+complex+subcellular+compartments.">Immunogold quantification of amino acids and proteins in complex subcellular compartments.</a> Nat. Protoc. 3, 144-152 (2008).</li><li>Orlov, I., 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=Live+cell+immunogold+labelling+of+RNA+polymerase.">Live cell immunogold labelling of RNA polymerase.</a> II. Sci. Rep. 5, 8324 (2015).</li><li>Fiala, G. J., Kaschek, D., Blumenthal, B., Reth, M., Timmer, J., Schamel, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-clustering+of+the+B+cell+antigen+receptor+demonstrated+by+mathematically+extended+electron+microscopy.">Pre-clustering of the B cell antigen receptor demonstrated by mathematically extended electron microscopy.</a> Front. Immunol. 4, 427 (2013).</li></ol>

The authors have nothing to disclose.

Studying the spatial distribution and stoichiometry of the membrane proteins, such as the EGFR, in whole cells provides important context information about its function and possible changes thereof11-14. It is challenging to study the distribution of monomers, dimers, and clusters directly on a subcellular level using commonly used biochemical methods, for which information is lost about the localization of the protein in the cell or differences between cells15. The labeling and microscopy methods described here allow the visualization of protein complexes on a length scale of a few nanometers so that its stoichiometry can be studied within the context of individual, intact cells4-7. This is not possible with (super resolution) light microscopy23 because its resolution is insufficient to distinguish molecules engaged in a protein complex. F&#246;rster Resonance Energy Transfer (FRET) is an ensemble averaging technique24, and does not necessarily detect the dimers and higher order clusters as a consequence of the short range of the energy transfer25. Proximity ligation assays26 do not actually measure distances and are thus not capable of distinguishing between a cellular region with a high protein density, inevitably leading to a number of detected proximities by random chance, from a cellular region with a similar protein density but containing protein complexes exhibiting distinct distances between the labels. The required high resolution to visualize the constituents of protein complexes is achieved by transmission electron microscopy (TEM). However, conventional TEM of whole cells is limited by the requirement of thin samples/sections slicing through the plasma membrane27, or plasma membrane ripping or breaking off28,29 resulting in randomly formed small pieces of membrane. The plasma membrane is thus not imaged as a whole, leading to a lack of possibly crucial cellular context information.
Other experimental challenges for the study of protein stoichiometry in cells arise from the applied protein labeling. Commonly used immunolabeling bears the difficulty that one-to-one stoichiometry between receptor and label can only be achieved with a monovalent probe; this is not the case when primary and secondary antibodies are required. To obtain information about the protein stoichiometry, it is required that a probe binds uniquely to one epitope on the receptor and has one binding site for a fluorescent probe or a high atomic number nanoparticle on the other side. Secondly, the precision achievable with antibody-labeling is restricted on account of their large size30 to 30 nm, so that a discrimination between neighboring single proteins, homodimers, or larger clusters is not feasible, at least not for receptors of the EGFR family. Much smaller specific labels are known in literature and can be applied even for intracellular labeling31 but those are not commonly used. The length of the whole label (EGF-biotin-streptavidin-QD) is of a similar dimension as the EGFR, and sufficiently small to be able to detect the dimer. Moreover, the described two-step labeling procedure avoids labeling induced receptor clustering.
Our method is not very difficult, not much more than fluorescence microscopy of labeled proteins. However, an experiment needs to be carried out with great care, since the number of steps adds up and an error in one of the steps may lead to a complete failure in the experiment. Handling the microchips is not more tedious than the handling of TEM grids but some training empty chips is recommended. ESEM-STEM of whole hydrated cells is probably the most difficult aspect, and requires at least a skilled operator and several days of practice in order to reach a resolution around 3 nm as needed to visualize the QDs. Radiation damage of the specimen under investigation presents a risk. The pressure and the temperature should be carefully watched to maintain a water layer. Moreover, the thickness of the water layer may vary between cells. It is advisable to monitor the presence of the water layer using the gaseous electron detector above the specimen, as described elsewhere6. Cellular regions should only be imaged once or twice to avoid damage.
A key limitation of the method is that high-resolution information about the ultrastructure is absent. Other techniques, for example, cryo TEM, are needed to study the protein structure, and the cellular ultrastructure15. Secondly, studying the dynamic interplay of a multiple of proteins in live cell in time-lapse microscope is not feasible on account of radiation damage, and requires state-of-the-art light microscopy23. Currently, our method is not capable of determining the absolute level of dimers since the labeling efficiency is unknown but we expect to add such data in future. It is nevertheless possible to tell if dimers are present or not. From the literature is it known, that even a labeling efficiency around 15% is enough to detect dimers with sufficient statistical significance32.
It is readily possible to study other membrane-bound receptors via their ligand, via biotinylated Fab fragments of specific antibodies, or via other small linkers7 using the described two-step labeling protocol. Since we have already demonstrated that two different colors (sizes) of QDs can be used, and gold nanoparticle labeling was used in prior work6, it seems feasible to label multiple protein species in the same experiment. The key challenge for the study of the stoichiometry of multiple protein species is ensuring a similar labeling efficiency for both species or at least a normalization of the obtained relative stoichiometry on the respective labeling efficiencies. Yet, the two different QDs used in this study do not seem to differ much in labeling efficiency (see Figures 2 and 4). The described method involving two-step labeling with QDs, and correlative fluorescence microscopy and ESEM-STEM presents a viable method to study the complex interplay of membrane proteins in the intact plasma membranes of whole cells.

A new approach was introduced recently, to image whole cells with labeled proteins in liquid state using STEM1-3, or correlative fluorescence microscopy and STEM4-7. This methodology is capable of studying the spatial distribution of membrane proteins and protein complexes including their stoichiometry at the single molecule level in the intact plasma membranes of whole cells. Here, we describe a protocol involving a two-step specific labeling of receptor proteins with fluorescent quantum dots (QDs)8, and correlative light microscopy and ESEM-STEM. As example, we studied the EGFR, a membrane protein that belongs to the protein kinase superfamily. Upon ligand binding, the receptor activates and then forms a dimer with another activated EGFR; the subsequent signal cascade drives cell proliferation9. Mutations leading to abnormal EGFR expression or activity play a central role in many types of cancer10. Studying the spatial arrangement of this membrane receptor in whole cells provides important context information about its function and possible changes thereof11-14. But it remains challenging to probe the distribution of EGFR monomers, dimers, and clusters directly on a (sub-)cellular level with biochemical- or conventional microscopy methods15. Our method is capable of visualizing EGFRs with a spatial resolution of a few nanometers such that the locations of proteins in a complex can be determined. Most importantly, the obtained information about the stoichiometric distribution relates to the native situation of the protein in the intact cell.
In order to achieve a short distance between the label and the receptor, EGFR was directly labeled via its ligand EGF, using a biotin-streptavidin bond to a QD. This label can be detected both with fluorescence- and electron microscopy. We have used this label for correlative imaging of COS7 cells in liquid enclosed in a microfluidic chamber previously1,16,17. However, on account of a multiple of streptavidin molecules per QD, this label may induce receptor clustering, leading to a low labeling efficiency or rather expensive experiments, and it is not possible to conclude on the stoichiometry of the protein under investigation. Label induced receptor clustering can be avoided altogether by employing the two-step labeling procedure presented here, resulting in a probe comprising biotinylated EGF to which only one streptavidin-quantum dot (STR-QD) is coupled. The cells are first incubated with biotinylated EGF, and then fixed. The fixation step determines the time point at which protein processes are stopped (depending on the speed of fixation). STR-QD is applied thereafter as second step of the labeling protocol. Clustering does not take place since dynamic membrane motion of the proteins is hindered once the proteins are fixed. Moreover, the STR-QD solution, which is the most expensive component of the experiment, can be applied in an optimized low concentration, and this second step of labeling is not time-crucial, i.e., the QD can be coupled in several minutes.
To study the stoichiometry of EGFR as distributed in whole cells, cellular samples were prepared on silicon microchips18. After applying the two-step QD labeling, and the recording of fluorescence microscopy images, the cells were rinsed with pure water, and imaged in hydrated state using ESEM-STEM19. The resulting correlative images provide information about the cellular EGFR distribution, and the stoichiometry in their natural context of hydrated cells. A similar method was used to study HER2 proteins in breast cancer cells in a recent study7.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Inverted fluorescence microscope</td>
			<td>Leica</td>
			<td>AF6000 + DMI6000B</td>
			<td></td>
		</tr>
		<tr>
			<td>ESEM</td>
			<td>FEI Company</td>
			<td>Quanta 400 FEG</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (1x) + GlutaMax + glucose + pyruvate</td>
			<td>Gibco</td>
			<td>31966-021</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS (1x) - Calcium chloride - Magnesium chlorid</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum, Performance Plus (FBS)</td>
			<td>Gibco</td>
			<td>16000-036</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellstripper</td>
			<td>Mediatech, Inc.</td>
			<td>25-056 Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>Water, chromasolv Plus for HPLC</td>
			<td>Sigma-Aldrich</td>
			<td>34877-2.5L</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS Roti-Stock 10x PBS</td>
			<td>Carl Roth</td>
			<td>1058.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, Rotisolv HPLC grade</td>
			<td>Carl Roth</td>
			<td>P076.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin Fraction V, biotin-free (BSA)</td>
			<td>Carl Roth</td>
			<td>O163.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin, from cold water fish skin</td>
			<td>Sigma-Aldrich</td>
			<td>G7041-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Carl Roth</td>
			<td>T873.1</td>
			<td></td>
		</tr>
		<tr>
			<td>shoul</td>
			<td>Molecular Probes</td>
			<td>E3477</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium teraborate decahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>S9640-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Sigma-Aldrich</td>
			<td>B6768-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Qdot 655 streptavidin conjugate</td>
			<td>Molecular Probes</td>
			<td>Q10121MP</td>
			<td></td>
		</tr>
		<tr>
			<td>Qdot 800 streptavidin conjugate</td>
			<td>Molecular Probes</td>
			<td>Q10173MP</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon microchips with silicon nitride membranes</td>
			<td>DENS Solutions</td>
			<td>Custom made</td>
			<td></td>
		</tr>
		<tr>
			<td>of 50 nm thickness and dimensions of 50 &#181;m x 0.40 mm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

studying the stoichiometry of epidermal growth factor receptor in intact cells using correlative microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

Figure 1 and 2 show representative images of QD655 labeled, membrane-bound EGFR visualized in intact, fully hydrated COS-7 cells. The DIC image in Figure 1 a gives an impression of the membrane topography of the cells, and the corresponding fluorescence image in Figure 1b depicts the distribution of EGFR after 3 min of EGF-Biotin incubation. Figure 1A and B are each stitched together from two images recorded with a 40X air objective. The EGF activated EGFR is distributed over the entire cellular surface. In most cells a slight enhancement of fluorescence (Figure 1B) can be seen at the cell edges, indicating a locally increased occurrence of EGFR20. Control experiments conducted using a similar protocol verified specific EGFR labeling (data not shown). These controls included: 1) a control labeling without previous incubation of the cells with EGF-biotin, i.e., only incubation with STR-QDs, and 2) an incubation with non-biotinylated EGF and STR-QDs.
The three cells marked with rectangles were further investigated with ESEM-STEM. The ESEM-STEM images depicted in Figure 1C-E show low magnification overviews of these cells. These transmission images reflect whole cells in hydrated state with a thin layer of water residing over the cell placed on a silicon microchip with silicon nitride (SiN) viewing window. The vacuum chamber in the microscope contained water vapor, and the balance between sample temperature and pressure was adjusted to maintain a thin layer of liquid over the cells. The individual cells can be easily recognized on account of their shape and location on the SiN membrane window. The low-magnification ESEM-STEM images reveal fine structures, such as filopodia, extending from the cell edge towards neighboring cells, and some structures inside the thinner cell regions. Thick central cellular regions including the nucleus appear white because transmission through the sample is not possible for electrons of the used energy (30 keV).
High-resolution ESEM-STEM images were recorded in the thinner, peripheral regions. The spatial resolution for ESEM-STEM of nanoparticles in the thin regions of cells in a thin liquid layer was determined in a previous study6 to amount to 3 nm. Four high-resolution images shown in Figure 2A-D were recorded at the locations of the small rectangles in Figure 1C-E. The used magnification was sufficient to discern individual QDs, appearing as bright, bullet-shaped rods, each bound to an individual EGFR. The QD655 has typical dimensions of 6 x 14 nm2.
Figure 2A (corresponding to the rectangular area indicated on the cell in Figure 1C) shows QD labels on a membrane fold diagonally crossing the image. This membrane structure has a higher EGFR density than the surrounding membrane regions. At several locations, two labels were at close proximity. Two examples with distances of 20 and 24 nm are indicated with arrowheads. These pairs of labels are interpreted as belonging to EGFR dimers. Figure 2B gives an example from the edge of a cell (Figure 1D, left rectangle), EGFR monomers and dimers can be seen as well. Figure 2C (Figure 1C, right rectangle) was recorded of a region with a lower fluorescence signal, i.e., lower EGFR density. Nevertheless, EGFR was also found here in dimers as well. In addition, two clusters of 10 or 11 EGFRs were present (see ellipses). Figure 2D shows another example of a membrane fold (Figure 1E) with smaller clusters including 5-6 EGFRs, in addition to several monomers and dimers.
As an example of the kind of information that can be obtained from this data, the pair correlation function21 g(x) was determined for all label positions in Figure 2. Note that this analysis is not part of this protocol and the procedure was described elsewhere6,7. g(x) is a measure of the chance of a particle to be found within a certain radial distance x from a reference particle. g(x) = 1 represents a random distribution, and a larger value is evidence for clustering. The positions of a total of 210 labels were automatically detected in the four images of Figure 2A-D, and g(x) was calculated using a bin size of 5 nm and a smoothing filter with a bandwidth of 10 nm. The g(x) curve (Figure 3) shows a preferred center-to-center QD distance of 25 nm. EGFRs are thus not randomly oriented but a significant fraction of them resides at this preferred distance. From an approximate molecular model6 (Figure 3 inset) we estimate that the center-to-center distance between the QD and the EGF binding pocket amounts to ~14 nm, and center-to-center distance between the two QDs attached to a EGFR dimer to be ~27 nm (this value is likely to vary by a few nanometers due to the flexibility of the linker). The preferred label distance is thus consistent with the expected label distance for the EGFR dimer within the precision of the method. The g(x) curve is larger than unity for distance of up to 300 nm, indicating the presence of clusters, consistent with the data. This analysis shows that the stoichiometry of the EGFR can be studied with our method.
Figure 4 shows a similar result as Figure 2, except that the labeling was performed with EGF-QD800, and a 63X oil immersion objective was used for the fluorescence image. This data confirms that these typical results are also found when the EGFR labeling is done with QDs, which emit in the near red spectrum. Figure 4A is the fluorescence image of a representative cell, Figure 4B shows the same cell in ESEM-STEM overview mode and Figure 4C is a 150,000 x magnification image, recorded at the upper membrane border of the cell (see rectangle in Figure 4B). Similar to Figure 2A and D, the images capture QD-labeled EGFR on a membrane fold and shows monomeric, dimeric, and clustered receptors. Note that the electron dense QD core appears somewhat smaller and rounder than the cores of QDs emitting at 655 nm, consistent with their smaller core size22 of ~5 nm.
<img alt="Figure 1" src="/files/ftp_upload/53186/53186fig1.jpg" /> 
Figure 1. Correlative DIC, fluorescence and ESEM-STEM of EGF-QD655 labeled EGFR on fully hydrated COS-7 cells. (A) DIC image of the cells grown on the SiN membrane window area, giving a topographic impression of the plasma membrane. (B) Fluorescence image showing cells on the centrally located SiN membrane window (marked by the dashed rectangle). (C-E) Three ESEM-STEM low magnification images from the cells marked with rectangles in A and B. These cell overview images serve to determine the precise location of subsequently recorded high-resolution images. The location of four high-resolution images (shown in Figure 2A-D) are marked with white rectangles. <a href="https://www.jove.com/files/ftp_upload/53186/53186fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53186/53186fig2.jpg" /> 
Figure 2. High-resolution ESEM-STEM images depicting membrane bound EGFRs. (A) Micrograph at 75,000X magnification of the area marked in Figure 1B. Many monomers are visible. Several dimers are indicated with the arrowheads. (B) and (C) images acquired at 50,000X magnification of the left, respectively right, membrane areas marked in Figure 1C. Clusters of EGFRs are outlined. (D) image recorded with 50,000X magnification at the location marked in Figure 1D. <a href="https://www.jove.com/files/ftp_upload/53186/53186fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53186/53186fig3.jpg" /> 
Figure 3. Pair correlation function g(x) as function of the radial distance x determined for the inter-particle distances of all 210 labels detected in Figure 2A-D. The peak at 25 nm indicates that a center-to-center QD distance has a much higher chance of occurring than random, whereby a g(x) = 1 indicates a random chance. The dashed line is a guide to the eye representing g(x) of a random distribution. The inset shows an approximate molecular model of the EGFR dimer with bound EGF and streptavidin coated QDs attached via biotin. The models of streptavidin, EGF and the EGFR were obtained from CPK models of the 1stp (streptavidin), 1EGF (EGF), 1NQL, 2JWA, 1M17, 1IVO and 2GS6 (EGFR) structures in the RCSB Protein Protein Databank, created by Jmol Version 12.2.15. The biotin model is as drawn in RCSB Ligand Explorer Version 1.0. <a href="https://www.jove.com/files/ftp_upload/53186/53186fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53186/53186fig4.jpg" /> 
Figure 4. Exemplary COS-7 cell labeled with EGF-QD800 and imaged with correlative fluorescence and ESEM-STEM. (A) Fluorescence image, (B) low magnification overview ESEM-STEM image. (C) High-resolution image recorded at the location of the rectangle in b at 150,000X magnification. Monomeric, dimeric, and clustered EGFRs are detected similar to labeling with EGF-QD655. <a href="https://www.jove.com/files/ftp_upload/53186/53186fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy at JoVE.com

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

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8.3K Views.  INM-Leibniz Institute for New Materials. This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level. 

Video: Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new devices for label-free separation and analysis of cells1. Such devices may use lateral displacement for continuous-flow separation, or receptor patterns that modulate adhesion to distinguish between different cell phenotypes or levels of receptor expression. Understanding the nature of cell rolling trajectories on receptor-patterned substrates is necessary for engineering of the substrates and design of such devices.
Here, we demonstrate a protocol for studying cell rolling trajectories on asymmetric receptor patterns that support cell rolling adhesion2. Well-defined, &mu;m-scale patterns of P-selectin receptors were fabricated using microcontact printing on gold-coated slides that were incorporated in a flow chamber. HL60 cells expressing the PSGL-1 ligand 3were flowed across a field of patterned lines and visualized on an inverted bright field microscope. The cells rolled and tracked along the inclined edges of the patterns, resulting in lateral deflection1. Each cell typically rolled for a certain distance along the pattern edges (defined as the edge tracking length), detached from the edge, and reattached to a downstream pattern. Although this detachment makes it difficult to track the entire trajectory of a cell from entrance to exit in the flow chamber, particle-tracking software was used to analyze and yield the rolling trajectories of the cells during the time when they were moving on a single receptor-patterned line. The trajectories were then examined to obtain distributions of cell rolling velocities and the edge tracking lengths for each cell for different patterns.
This protocol is useful for quantifying cell rolling trajectories on receptor patterns and relating these to engineering parameters such as pattern angle and shear stress. Such data will be useful for design of microfluidic devices for label-free cell separation and analysis.

We describe a protocol to observe and analyze cell rolling trajectories on asymmetric receptor-patterned substrates. The resulting data are useful for engineering of receptor-patterned substrates for label-free cell separation and analysis.

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new ...

Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata are separated from their axon terminals due to skeletal growth of the enlarging organism (Weiss 1941; Gray, Hukkanen et al. 1992). This mechanotransduction induces a secondary mode of neuronal growth capable of accommodating continual elongation of the axon (Bray 1984; Heidemann and Buxbaum 1994; Heidemann, Lamoureux et al. 1995; Pfister, Iwata et al. 2004).
Axon Stretch Growth (ASG) is conceivably a central factor in the maturation of short embryonic processes into the long nerves and white matter tracts characteristic of the adult nervous system. To study ASG in vitro, we engineered bioreactors to apply tension to the short axonal processes of neuronal cultures (Loverde, Ozoka et al. 2011). Here, we detail the methods we use to prepare bioreactors and conduct ASG. First, within each stretching lane of the bioreactor, neurons are plated upon a micro-manipulated towing substrate. Next, neurons regenerate their axonal processes, via growth cone extension, onto a stationary substrate. Finally, stretch growth is performed by towing the plated cell bodies away from the axon terminals adhered to the stationary substrate; recapitulating skeletal growth after growth cone extension.
Previous work has shown that ASG of embryonic rat dorsal root ganglia neurons are capable of unprecedented growth rates up to 10mm/day, reaching lengths of up to 10cm; while concurrently resulting in increased axonal diameters (Smith, Wolf et al. 2001; Pfister, Iwata et al. 2004; Pfister, Bonislawski et al. 2006; Pfister, Iwata et al. 2006; Smith 2009). This is in dramatic contrast to regenerative growth cone extension (in absence of mechanical stimuli) where growth rates average 1mm/day with successful regeneration limited to lengths of less than 3cm (Fu and Gordon 1997; Pfister, Gordon et al. 2011). Accordingly, further study of ASG may help to reveal dysregulated growth mechanisms that limit regeneration in the absence of mechanical stimuli.

A unique tissue engineering method was developed to elongate numerous nerve fibers in culture by recapitulating axon stretch growth; a form of nervous system growth whereby nerves elongate in conjunction with growth of the enlarging body.

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata ...

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. 
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. 
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging2, due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-13-7, the resolution afforded by live-cell microscopy is limited (~ 200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target.
In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to maintain cellular homeostasis. Puncture injury with glass needles provides a direct method to trigger a rapid epidermal wound response that activates wound transcriptional reporters, which can be visualized by a localized reporter signal in living embryos or larvae. Puncture or laser injury also provides signals that promote the recruitment of hemocytes to the wound site. Surprisingly, severe (through and through) puncture injury in late stage embryos only rarely disrupts normal embryonic development, as greater than 90% of such wounded embryos survive to adulthood when embryos are injected in an oil medium that minimizes immediate leakage of hemolymph from puncture sites. The wound procedure does require micromanipulation of the Drosophila embryos, including manual alignment of the embryos on agar plates and transfer of the aligned embryos to microscope slides. The Drosophila epidermal wound response assay provides a quick system to test the genetic requirements of a variety of biological functions that promote wound healing, as well as a way to screen for potential chemical compounds that promote wound healing. The short life cycle and easy culturing routine make Drosophila a powerful model organism. Drosophila clean wound healing appears to coordinate the epidermal regenerative response, with the innate immune response, in ways that are still under investigation, which provides an excellent system to find conserved regulatory mechanisms common to Drosophila and mammalian epidermal wounding.

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The embryonic epidermis of very late stage Drosophila embryos provides an in vivo system for rapid puncture wound response analysis and can be combined with genetic manipulations or chemical microinjection treatments to advance studies in wound healing for translation into mammalian models.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to ...

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating&#160;poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth.  This system incorporates mechanical, topographical, ...

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone fusion. Large doses of highly potent biological agents are required due to growth factor instability as a result of rapid enzymatic degradation as well as carrier inefficiencies in localizing sufficient amounts of growth factor at implant sites. Hence, strategies that prolong the stability of growth factors such as BMP-2/NELL-1, and control their release could actually lower their efficacious dose and thus reduce the need for larger doses during future bone regeneration surgeries. This in turn will reduce side effects and growth factor costs. Self-assembled PECs have been fabricated to provide better control of BMP-2/NELL-1 delivery via heparin binding and further potentiate growth factor bioactivity by enhancing in vivo stability. Here we illustrate the simplicity of PEC fabrication which aids in the delivery of a variety of growth factors during reconstructive bone surgeries.

Self-assembled polyelectrolyte complexes (PEC) fabricated from heparin and protamine were deposited on alginate beads to entrap and regulate the release of osteogenic growth factors. This delivery strategy enables a 20-fold reduction of BMP-2 dose in spinal fusion applications. This article illustrates the benefits and fabrication of PECs.

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone ...

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein (HDL)-mimicking nanoparticles (NPs). HDLs are endogenous NPs and have been explored as vehicles for the delivery of therapeutic agents. Various methods have been developed to prepare HDL-mimicking NPs. However, they are generally complicated, time consuming, and difficult for industrial scale-up. In this study, one-step homogenization was used to mix the excipients and form the prototype NPs. NGF is a water-soluble protein of 26 kDa. To facilitate the encapsulation of NGF into the lipid environment of HDL-mimicking NPs, protamine USP was used to form an ion-pair complex with NGF to neutralize the charges on the NGF surface. The NGF/protamine complex was then introduced into the prototype NPs. Apolipoprotein A-I was finally coated on the surface of the NPs. NGF HDL-mimicking NPs showed preferable properties in terms of particle size, size distribution, entrapment efficiency, in vitro release, bioactivity, and biodistribution. With the careful design and exploration of homogenization in HDL-mimicking NPs, the procedure was greatly simplified, and the NPs were made scalable. Moreover, various challenges, such as separating unloaded NGF from the NPs, conducting reliable in vitro release studies, and measuring the bioactivity of the NPs, were overcome.

Simple homogenization was used to prepare novel, high-density, lipoprotein-mimicking nanoparticles to encapsulate nerve growth factor. Challenges, detailed protocols for nanoparticle preparation, in vitro characterization, and in vivo studies are described in this article.

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein ...