The authors have nothing to disclose.

1. Nanostructure Fabrication
<ol>
	<li>Choose 25 mm diameter glass coverslips with an approximate thickness of 170 &#181;m (No. 1.5) as substrates for nanofabrication.</li>
	<li>Immerse the coverslips in piranha solution (3:1 ratio of sulphuric acid and hydrogen peroxide) for at least 6 hr. Wash the piranha soaked coverslip with copious amount of ultrapure 18.2 M&#8486; deionized distilled water (DDW). 
	CAUTION: Piranha acid reacts violently with organic materials and must be handled with extreme care.</li>
	<li>Deposit 10 nm chromium thin film on the coverslips by e-beam evaporation to avoid charge effects during the patterning and imaging of nanostructures.</li>
	<li>Spin the first layer of bilayer resist consisting of ethyl lactate methyl methacrylate (MMA_EL6) copolymer at 2,000 rpm for 45 sec and then bake at 150 &#176;C. Spin the second layer of poly-methyl methacrylate (950PMMA_A2) at 3,000 rpm for 45 sec then bake at 180 &#176;C.</li>
	<li>Pattern the bilayer resist using the electron beam lithography (EBL) at 25 kV with an area dose of 300 &#181;C/cm2. Develop in isopropyl alcohol (IPA)/methyl isobutyl ketone (MIBK): 2/1 and rinse in IPA.</li>
	<li>Deposit Ti (5 nm) / Au (80 nm) film on the substrate using a e-beam evaporator.</li>
	<li>Following the gold deposition, lift off the copolymer bilayer resist by soaking the substrate in acetone for 4 hr.</li>
	<li>Inspect the substrate using the scanning electron microscope (SEM) to confirm nanostructure shape and size, remove the remaining chromium from the substrate via wet etch using CR-7 etchant for 60 sec at RT and then rinse in DDW.</li>
	<li>Design a center-to-center array spacing of 33 &#181;m to leave room for cell imaging between arrays. Pattern the nanostructures in 20 x 20 pattern for each array with a pitch of 300 nm using an e-beam writer. Each chip contains 300 arrays with a typical nanostructure dimension of 80&#177;2.5 nm height and 70&#177;2.5 nm diameter.</li>
	<li>Inspect a subset of arrays using the atomic force microscope (AFM) for the verification of size and uniformity.</li>
	<li>Attach a support ring, typically silicon, to the back of the coverslip using a UV curing epoxy.</li>
</ol>
2. Chip Cleaning and Application of Self-assembled Monolayer
<ol>
	<li>For cleaning and regenerating the chips, plasma ash at a power of 40 W in a 300 mTorr mixture of 5% hydrogen, 95% argon for 45 sec after cleaning the chamber for 5 min under the same conditions.</li>
	<li>Functionalize the gold nanostructures immediately after plasma ashing by immersing the chip in a two-component ethanolic thiol solution consisting of a 3:1 ratio of SH-(CH2)8-EG3-OH (SPO) and a component with either an amine or carboxyl functional group, namely, SH-(CH2)11-EG3-NH2 (SPN) or SH-(CH2)11-EG3-COOH (SPC).</li>
	<li>Leave the chip in the thiol solution O/N&#160;to form a self-assembled monolayer (SAM).</li>
	<li>Rinse the chip with ethanol and dry with nitrogen gas.</li>
	<li>If needed, store the functionalized chip for up to 2 weeks at 4 &#176;C.</li>
	<li>When ready for use react the SPN or SPC component with the ligand using a chemistry depending on the ligand of choice (see below). 
	Note: The chips can be regenerated and re-functionalized dozens of times. A given chip can be used for periods that range from 6 months to over a year. The spectra measured on a given array are reliably reproduced after repeated regenerations by plasma ashing, followed by biofunctionalization.29</li>
</ol>
3. Surface Functionalization and Kinetic Characterization
Note: Use the functionalized chip in the commercial SPR instrument to characterize the kinetic rate constants between the ligand and the analyte, as well as to study the resistance of SAM to non-specific binding. There is a wide range of flow rates and microfluidic designs that allow for efficient surface functionalization. Since we have a commercially available SPR we standardized around its recommended flow rates. We note that these flow rates are typical of all SPR instruments and so are not restrictive. The SPR instrument is not a necessity since all functionalization can be done directly on the LSPR chip, but it did reduce our work load because it is a multiplexed instrument whereas our LSPR microfluidic setup is not.
<ol>
	<li>Functionalize a commercial bare gold chip with the SAM as described in Section 2.</li>
	<li>If using an SPC based SAM, activate the carboxyl group with a 1:1 mixture of 133 mM of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 33 mM of N-hydroxysuccinimide (NHS) in DDW for 10 min.</li>
	<li>Conjugate the activated carboxyl group with the antibody/ligand of interest for 300 sec using a flow rate of 30 &#181;l/min. Prepare the ligands in pH 6 phosphate buffer, typically, but this can vary depending on the molecule.</li>
	<li>After the ligand conjugation, flow 0.1 M ethanolamine in phosphate-buffered saline (PBS) as a deactivator step for 300 sec at a rate of 30 &#181;l/min. Ethanolamine helps in minimizing the non-specific binding.</li>
	<li>Introduce the analyte of interest at a flow rate of 100 &#181;l/min using a range of concentrations and calculate the kinetic rate constants using a kinetic analysis software.</li>
	<li>If non-specific binding is problematic, increase the ratio of SPO to SPC or SPN.</li>
</ol>
4. LSPR General Settings
<ol>
	<li>Microscope settings:
	<ol>
		<li>Use a 100 W Halogen lamp to Koehler illuminate the sample. Use a long pass filter (typically 593 nm cutoff) in the light path to eliminate wavelengths that do not contribute to the resonance shift (Figure 2).</li>
		<li>For the LSPRi data collection, use an inverted microscope with a 40X oil immersion objective (1.4 NA) and a thermoelectrically cooled 16 bit CCD camera.</li>
		<li>Place a beam splitter at the output port of the microscope to obtain Imagery and spectra simultaneously.</li>
		<li>Set the temperature controlled microscope stage to 37 &#176;C and equilibrate for 4 hr.</li>
		<li>Incorporate an additional incubation assembly on the microscope to regulate the CO2 concentration and humidity at 5% and 95%, respectively.</li>
	</ol>
	</li>
	<li>Chip preparation and mounting:
	<ol>
		<li>Functionalize the LSPR chip as described in Section 2 with the optimal two-component SAM ratios determined from the SPR experiments.</li>
		<li>Load the chip within a custom made microfluidic holder as follows. Place the chip on an aluminum bottom piece. Sandwich the chip between this bottom piece and a silicone gasket and a clear plastic top piece. Use 4 screws to clamp the assembly.</li>
		<li>For a typical SPC-based thiol application, drop coat 300 &#181;l a 1:1 mixture of 133 mM of EDC and 33 mM of NHS in DDW to activate the carboxyl groups of the SPC thiol component.</li>
		<li>Wait for 10 min&#160;and manually rinse the surface with 10 mM PBS.</li>
		<li>Conjugate the activated carboxyl group with the ligand (typically an antibody or antibody fragment) of interest by drop coating 300 &#181;l of ligand solution.</li>
		<li>Wait for 30 min and manually rinse with 10 mM PBS.</li>
		<li>Drop coat 300 &#181;l of 0.1 M ethanolamine in PBS on the chip to minimize nonspecific binding. Wait for 10 min.</li>
		<li>Wash the ethanolamine with PBS containing 0.005% Tween 20 (PBS-T20).</li>
		<li>Place a quartz piece above the chip to reduce fluctuations in the data related to a changing meniscus.</li>
		<li>Keep the chip wet with PBS-T20 buffer while mounting on microscope.</li>
		<li>Place the LSPR chip assembly firmly into the heated stage sample holder and attach microfluidic tubing.</li>
		<li>Attach the microfluidics tubing to the assembly and flow buffer (or serum free media for cell studies) until a steady state is reached.</li>
		<li>Allow the assembly and microscope to equilibrate for at least 2 hr.</li>
		<li>Align the chip using joystick so that the central array is aligned with the fiber optic for spectroscopy. Spectroscopic data is taken using a spectrometer and spectral analysis software.</li>
		<li>Keep arrays in focus throughout the experiment by using software autofocus, Zeiss Definite Focus or equivalent autofocus device.</li>
	</ol>
	</li>
</ol>
5. LSPR Imaging of Anti-c-myc Secretions from 9E10 Hybridoma Cells
Note: The hybridoma cell line used for this study express anti-c-myc antibody constitutively and hence do not require a chemical trigger
<ol>
	<li>Functionalize the nanostructures with c-myc peptide. This has a KD value of 1.77 nM for the anti-c-myc antibodies secreted by clone 9E10 hybridoma cells.</li>
	<li>Culture the hybridoma cells in complete growth medium with 10% fetal bovine serum (FBS) and 1% antibiotic in a T75 flask at 37 &#176;C under 5% CO2. Maintain a cell density of 4 &#215;105 cells/ml.</li>
	<li>For the cell secretion studies, pellet cells from the T75 flask by centrifugation, wash twice with RPMI-1640 serum free media (SFM) to remove the secreted antibodies and adjust the cell density to 4 &#215;106 cells/ml.</li>
	<li>Harvest the cells and test for viability before introducing them on to the LSPR chips.</li>
	<li>Introduce 50 &#181;l of the cell solution manually onto the LSPR chips with a micropipette. After few minutes 25 to 50 cells adhere to the surface of the LSPR chips.</li>
	<li>Wash away the remaining cells in solution with fresh SFM using the microfluidic perfusion system.</li>
	<li>Select the LSPR arrays for imaging which are close, within 10 &#956;m, but not overlapping with the cells.</li>
	<li>To ensure that the signal is specific to the secreted anti-c-myc antibodies introduce the cell culture media with and without the antibodies present as well as with the antibodies but with their binding sites blocked by the presence of a saturating concentration of c-myc peptide in the solution.</li>
	<li>Calibrate the sensors at the end of each run with a saturating solution of anti-c-myc antibodies (250 nM). This helps in normalizing the response of the sensors and determine fractional occupancy based on the biofunctionalization profile of each run.</li>
	<li>Correct the drift in the X and Y direction using image alignment software.</li>
</ol>

<ol>
	<li>Ludwig, A. -. K., Giebel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes:+Small+vesicles+participating+in+intercellular+communication.">Exosomes: Small vesicles participating in intercellular communication.</a> The International Journal of Biochemistry &amp; Cell Biology. 44, 11-15 (2012).</li><li>Friedl, P., Gilmour, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+cell+migration+in+morphogenesis,+regeneration+and+cancer.">Collective cell migration in morphogenesis, regeneration and cancer.</a> Nature Reviews Molecular Cell Biology. 10, 445-457 (2009).</li><li>Letterio, J. J., Roberts, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+immune+responses+by+TGF-beta.">Regulation of immune responses by TGF-beta.</a> Annual Review of Immunology. 16, 137-161 (1998).</li><li>Werner, S., Grose, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+wound+healing+by+growth+factors+and+cytokines.">Regulation of wound healing by growth factors and cytokines.</a> Physiological Reviews. 83, 835-870 (2003).</li><li>Werner, S., Krieg, T., Smola, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keratinocyte-fibroblast+interactions+in+wound+healing.">Keratinocyte-fibroblast interactions in wound healing.</a> Journal of Investigative Dermatology. 127, 998-1008 (2007).</li><li>Bailey, R. C., Kwong, G. A., Radu, C. G., Witte, O. N., Heath, 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=DNA-encoded+antibody+libraries:+A+unified+platform+for+multiplexed+cell+sorting+and+detection+of+genes+and+proteins.">DNA-encoded antibody libraries: A unified platform for multiplexed cell sorting and detection of genes and proteins.</a> Journal of the American Chemical Society. 129, 1959-1967 (2007).</li><li>Gazagne, 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=A+Fluorospot+assay+to+detect+single+T+lymphocytes+simultaneously+producing+multiple+cytokines.">A Fluorospot assay to detect single T lymphocytes simultaneously producing multiple cytokines.</a> Journal of Immunological Methods. 283, 91-98 (2003).</li><li>Han, Q., 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=Polyfunctional+responses+by+human+T+cells+result+from+sequential+release+of+cytokines.">Polyfunctional responses by human T cells result from sequential release of cytokines.</a> Proceedings of the National Academy of Sciences of the United States of America. 109, 1607-1612 (2012).</li><li>Han, Q., Bradshaw, E. M., Nilsson, B., Hafler, D. A., Love, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multidimensional+analysis+of+the+frequencies+and+rates+of+cytokine+secretion+from+single+cells+by+quantitative+microengraving.">Multidimensional analysis of the frequencies and rates of cytokine secretion from single cells by quantitative microengraving.</a> Lab on a Chip. 10, 1391-1400 (2010).</li><li>Ma, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+clinical+microchip+for+evaluation+of+single+immune+cells+reveals+high+functional+heterogeneity+in+phenotypically+similar+T+cells.">A clinical microchip for evaluation of single immune cells reveals high functional heterogeneity in phenotypically similar T cells.</a> Nature Medicine. 17, 738-743 (2011).</li><li>Shirasaki, Y., 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=Real-time+single-cell+imaging+of+protein+secretion.">Real-time single-cell imaging of protein secretion.</a> Scientific Reports. 4, (2014).</li><li>Milgram, 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=On+chip+real+time+monitoring+of+B-cells+hybridoma+secretion+of+immunoglobulin.">On chip real time monitoring of B-cells hybridoma secretion of immunoglobulin.</a> Biosensors and Bioelectronics. 26, 2728-2732 (2011).</li><li>Abbas, A., Linman, M. J., Cheng, Q. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+trends+in+instrumental+design+for+surface+plasmon+resonance-based+biosensors.">New trends in instrumental design for surface plasmon resonance-based biosensors.</a> Biosensors &amp; Bioelectronics. 26, 1815-1824 (2011).</li><li>Ermakova, 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=Detection+of+a+Few+Metallo-Protein+Molecules+Using+Color+Centers+in+Nanodiamonds.">Detection of a Few Metallo-Protein Molecules Using Color Centers in Nanodiamonds.</a> Nano Letters. 13, 3305-3309 (2013).</li><li>Haes, A. J., Van Duyne, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+nanoscale+optical+blosensor:+Sensitivity+and+selectivity+of+an+approach+based+on+the+localized+surface+plasmon+resonance+spectroscopy+of+triangular+silver+nanoparticles.">A nanoscale optical blosensor: Sensitivity and selectivity of an approach based on the localized surface plasmon resonance spectroscopy of triangular silver nanoparticles.</a> Journal of the American Chemical Society. 124, 10596-10604 (2002).</li><li>Horowitz, V. R., Aleman, B. J., Christle, D. J., Cleland, A. N., Awschalom, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+spin+resonance+of+nitrogen-vacancy+centers+in+optically+trapped+nanodiamonds.">Electron spin resonance of nitrogen-vacancy centers in optically trapped nanodiamonds.</a> Proceedings of the National Academy of Sciences of the United States of America. 109, 13493-13497 (2012).</li><li>Sepulveda, B., Angelome, P. C., Lechuga, L. M., Liz-Marzan, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LSPR-based+nanobiosensors.">LSPR-based nanobiosensors.</a> Nano Today. 4, 244-251 (2009).</li><li>Barbillon, G., 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=Biological+and+chemical+gold+nanosensors+based+on+localized+surface+plasmon+resonance.">Biological and chemical gold nanosensors based on localized surface plasmon resonance.</a> Gold Bulletin. 40, 240-244 (2007).</li><li>Endo, 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=Multiple+label-free+detection+of+antigen-antibody+reaction+using+localized+surface+plasmon+resonance-based+core-shell+structured+nanoparticle+layer+nanochip.">Multiple label-free detection of antigen-antibody reaction using localized surface plasmon resonance-based core-shell structured nanoparticle layer nanochip.</a> Analytical Chemistry. 78, 6465-6475 (2006).</li><li>Endo, T., Kerman, K., Nagatani, N., Takamura, Y., Tamiya, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+detection+of+peptide+nucleic+acid-DNA+hybridization+using+localized+surface+plasmon+resonance+based+optical+biosensor.">Label-free detection of peptide nucleic acid-DNA hybridization using localized surface plasmon resonance based optical biosensor.</a> Analytical Chemistry. 77, 6976-6984 (2005).</li><li>Haes, A. J., Hall, W. P., Chang, L., Klein, W. L., Van Duyne, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+localized+surface+plasmon+resonance+biosensor:+First+steps+toward+an+assay+for+Alzheimer's+disease.">A localized surface plasmon resonance biosensor: First steps toward an assay for Alzheimer's disease.</a> Nano Letters. 4, 1029-1034 (2004).</li><li>Jonsson, M. P., Jonsson, P., Dahlin, A. B., Hook, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supported+lipid+bilayer+formation+and+lipid-membrane-mediated+biorecognition+reactions+studied+with+a+new+nanoplasmonic+sensor+template.">Supported lipid bilayer formation and lipid-membrane-mediated biorecognition reactions studied with a new nanoplasmonic sensor template.</a> Nano Letters. 7, 3462-3468 (2007).</li><li>Park, J. 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=A+regeneratable,+label-free,+localized+surface+plasmon+resonance+(LSPR)+aptasensor+for+the+detection+of+ochratoxin+A..">A regeneratable, label-free, localized surface plasmon resonance (LSPR) aptasensor for the detection of ochratoxin A..</a> Biosensors &amp; Bioelectronics. 59, 321-327 (2014).</li><li>Mayer, K. M., Hao, F., Lee, S., Nordlander, P., Hafner, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+molecule+immunoassay+by+localized+surface+plasmon+resonance.">A single molecule immunoassay by localized surface plasmon resonance.</a> Nanotechnology. 21, (2010).</li><li>Endo, T., Yamamura, S., Kerman, K., Tamiya, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+cell-based+assay+using+localized+surface+plasmon+resonance+biosensor.">Label-free cell-based assay using localized surface plasmon resonance biosensor.</a> Analytica Chimica Acta. 614, 182-189 (2008).</li><li>Huang, Y. X., Cai, D., Chen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-+and+Nanotechnologies+for+Study+of+Cell+Secretion.">Micro- and Nanotechnologies for Study of Cell Secretion.</a> Analytical Chemistry. 83, 4393-4406 (2011).</li><li>Oh, B. R., 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=Integrated+Nanoplasmonic+Sensing+for+Cellular+Functional+Immunoanalysis+Using+Human+Blood.">Integrated Nanoplasmonic Sensing for Cellular Functional Immunoanalysis Using Human Blood.</a> ACS Nano. 8, 2667-2676 (2014).</li><li>Raphael, M. P., Christodoulides, J. A., Delehanty, J. B., Long, J. P., Byers, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Imaging+of+Protein+Secretions+from+Single+Cells+in+Real+Time.">Quantitative Imaging of Protein Secretions from Single Cells in Real Time.</a> Biophysical Journal. 105, 602-608 (2013).</li><li>Raphael, M. 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=A+New+Methodology+for+Quantitative+LSPR+Biosensing+and+Imaging.">A New Methodology for Quantitative LSPR Biosensing and Imaging.</a> Analytical Chemistry. 84, 1367-1373 (2011).</li><li>Raphael, M. 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=Quantitative+LSPR+imaging+for+biosensing+with+single+nanostructure+resolution.">Quantitative LSPR imaging for biosensing with single nanostructure resolution.</a> Biophysical Journal. 104, 30-36 (2013).</li><li>Raphael, M. 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=A+new+methodology+for+quantitative+LSPR+biosensing+and+imaging.">A new methodology for quantitative LSPR biosensing and imaging.</a> Analytical Chemistry. 84, 1367-1373 (2012).</li><li>Henn, A. D., 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=Modulation+of+single-cell+IgG+secretion+frequency+and+rates+in+human+memory+B+cells+by+CpG+DNA,+CD40L,+IL-21,+and+cell+division.">Modulation of single-cell IgG secretion frequency and rates in human memory B cells by CpG DNA, CD40L, IL-21, and cell division.</a> Journal of Immunology. 183, 3177-3187 (2009).</li><li>Bramswig, K. 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=Soluble+Carcinoembryonic+Antigen+Activates+Endothelial+Cells+and+Tumor+Angiogenesis.">Soluble Carcinoembryonic Antigen Activates Endothelial Cells and Tumor Angiogenesis.</a> Cancer Research. 73, 6584-6596 (2013).</li></ol>

We thank George Anderson for helpful comments and discussions. This work was supported by the Naval Research Laboratory&#8217;s Institute for Nanoscience and the National Research Council Research Associateship Award.

The LSPR imaging technique described in this work has numerous advantages over more traditional methodologies for detecting cell secretions. First, the time resolution of our technique is on the order of seconds whereas the commercial alternative, an immunosandwhich assay known as EliSpot, has a typical time resolution of 2 to 3 days.7,32 As a result we were able to resolve sudden changes in the rate of protein secretion, such as that shown in Figure 6. Second, having arrays distributed over the chip allows for the secreted signal to be tracked in space and time which enables more rigorous comparisons to diffusion-based models of cell secretion. In addition, arrays like the control array shown in Figure 6 can be used to subtract out global changes in the image that typically arise from instrumental factors such as focus drift. Third, our technique requires no modification of the cells. If desired, the experiment can incorporate commonly used tags such as fluorescent proteins, but if there is concern that such tags may negatively affect cell viability or homeostasis the label-free nature of our approach does not require them. Fourth, using the spectroscopic data we have demonstrated that quantitative information regarding the fractional occupancy of surface bound ligands can be calculated.
There are numerous alternative methods to EBL for fabricating metallic nanoparticles. However, we have found that the EBL provides considerable flexibility for optimizing nanostructure and array dimensions to best suit the optics and the cells under investigation. Also critical is the fact that the chips can be readily regenerated by plasma ashing. In this way, a typical chip can be used dozens of times. Biofunctionalization details must be modified for the specific application. The protocol presented here conjugated the surface with relatively small c-myc peptide ligands. Larger ligands such as whole antibodies typically require more spacing and thus a higher SPO to SPN/SPC ratio. Regardless, a well formed SAM layer is essential for preventing non-specific binding in live-cell experiments. In general, larger molecular weight analytes are more readily detected by LSPR. Thus, in its single-cell manifestation, this technique may not be appropriate for detecting the secretion of small proteins, such as cytokines. 
The current setup has been used for studying individual non-adherent cells. There are significant number of secreted signaling proteins and vesicles to which the results reported in this work are directly applicable. For example carcinoembryonic antigen (CEA) which for decades now has been a diagnostic marker for cancer. Colon cancer cells are known to secrete CEA at the rates of thousands of molecules/cell/hr and the molecular weight is 180 kDa which exceeds that of IgG antibodies. CEA is believed to be involved in autocrine and paracrine signaling pathways but the spatio-temporal nature of these secretions have never been measured. Our technique can directly address these signaling questions. An extension of this work will be to measure the spatio-temporal nature of CEA secretion from single cells.33 Future work will also focus on integrating LSPRi with two and three dimensional cell cultures of adherent cells. By incorporating multiplexed arrays capable of detecting a number of secreted proteins in parallel, this technique has the potential to open a new window into cell secretions and how they influence neighboring cells.

Inter-cellular communication is crucial for the regulation of many physiological activities both inside and outside the cell. A variety of proteins and vesicles can be secreted that subsequently trigger complex cellular processes such as differentiation, wound healing, immune response, migration, and proliferation.1-5 Malfunctions of inter-cellular signaling pathways have been implicated in numerous disorders including cancer, atherosclerosis, and diabetes, to name a few.
The optimal cell secretion assay should be capable of spatially and temporally mapping the secreted protein of interest without disrupting the relevant signaling pathways. In this way causal relationships between the concentration profiles and the response of the receiving cells can be inferred. Unfortunately, many of the most commonly used fluorescent-based techniques do not meet these criteria. Fluorescent fusion proteins can be used to tag the analyte within the cell but can disrupt the secretory pathway, or if secreted, results in a diffuse glow outside the cell which is difficult to quantify. Fluorescent immunosandwich-based assays are the most commonly used techniques for detecting cellular secretions but typically require the isolation of individual cells.6-11 In addition, the introduction of the sensing antibody typically halts or ends the experiment and the size of the antibody labels, 150 kDa for IgG, is an impediment to downstream signaling.
Because of these roadblocks it is preferable that a label-free technique be utilized to image protein secretions and amongst existing label-free technologies, surface plasmon resonance (SPR) and localized surface plasmon resonance (LSPR) sensors are excellent candidates.12-17 These sensors have been widely used for analyte binding studies of proteins, exosomes and other biomarkers.18-24 In the case of LSPR, the plasmonic nanostructures can be patterned lithographically onto glass coverslips and excited using visible light via standard wide-field microscopy configurations. Due to their nanoscale footprint, the majority of the glass substrate is available for common imaging techniques such as bright-field and fluorescence microscopy making these probes well suited for integration with live-cell microscopy.25-28 We have demonstrated the real-time measurement of antibody secretions from hybridoma cells using functionalized gold plasmonic nanostructures with spatial and temporal resolutions of 225 msec and 10 &#181;m, respectively. The basic chip configuration is illustrated in Figure 1.28 The output light path of the microscope is split between a CCD camera used for imagery and a fiber-optically coupled spectrometer for the quantitative determination of fractional occupancy of a given array of nanostructures (Figure 2).
The protocol presented in this paper describes the experimental design for the real-time measurement of single cell secretions while simultaneously monitoring the response of the cells using the standard bright-field microscopy. The multidisciplinary approach includes the fabrication of nanostructures, functionalization of the nanostructures for the high affinity binding of analytes, surface optimization for both minimizing non-specific binding and characterizing kinetic rate constants using a commercial Surface Plasmon Resonance (SPR) instrument, integration of cell lines onto the substrate, and the analysis of imagery and spectral data. We anticipate this technique to be an enabling technology for the spatio-temporal mapping of cell secretions and their causal relationships with receiving cells.

<table><tbody><tr><td>25&amp;#160;mm diameter glass coverslips</td><td>Bioscience Tools&amp;nbsp;</td><td>CSHP-No1.5-25</td><td>&amp;nbsp; 170&amp;plusmn;5 &amp;micro;m is optimal</td></tr><tr><td>Poly-methyl methacrylate</td><td>Microchem</td><td>PMMA 950 A4</td><td></td></tr><tr><td>Ethyl lactate methyl metacrylate</td><td>Microchem</td><td>MMA EL6</td><td></td></tr><tr><td>Electron beam evaporator</td><td>Temescal</td><td>FC-2000</td><td></td></tr><tr><td>Electron beam lithography</td><td>Raith</td><td>Series 150</td><td></td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>459836</td><td></td></tr><tr><td>Acetone</td><td>Sigma-Aldrich</td><td>320110</td><td></td></tr><tr><td>CR-7 chromium etchant</td><td>Cyantek</td><td>CR-7</td><td></td></tr><tr><td>Scanning electron microscope</td><td>Zeiss</td><td>Ultra 55</td><td></td></tr><tr><td>Atomic force microscope</td><td>Veeco</td><td>Nanoscope III</td><td></td></tr><tr><td>Plasma ashing system</td><td>Technics</td><td>Series 85 RIE</td><td></td></tr><tr><td>SH-(CH&lt;sub&gt;2&lt;/sub&gt;)&lt;sub&gt;8&lt;/sub&gt;-EG&lt;sub&gt;3&lt;/sub&gt;-OH (SPO)</td><td>Prochimia</td><td>TH 001-m8.n3-0.2</td><td></td></tr><tr><td>SH-(CH&lt;sub&gt;2&lt;/sub&gt;)&lt;sub&gt;11&lt;/sub&gt;-EG&lt;sub&gt;3&lt;/sub&gt;-COOH (SPC)</td><td>Prochimia</td><td>TH 003m11n3-0.1</td><td></td></tr><tr><td>SH-(CH&lt;sub&gt;2&lt;/sub&gt;)&lt;sub&gt;11&lt;/sub&gt;-EG&lt;sub&gt;3&lt;/sub&gt;-NH&lt;sub&gt;2&lt;/sub&gt; (SPN)</td><td>Prochimia</td><td>TH 002-m11.n3-0.2</td><td></td></tr><tr><td>Surface plasmon resonance system</td><td>Biorad</td><td>XPR36</td><td></td></tr><tr><td>Bare gold chip</td><td>Biorad</td><td>GLC chip</td><td>Plasma ashed to remove the monolayer</td></tr><tr><td>1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide</td><td>Thermo</td><td>22980</td><td></td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-hydroxysuccinimide (NHS)</td><td>Thermo</td><td>24510</td><td></td></tr><tr><td>Pentylamine-Biotin&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Thermo</td><td>21345</td><td></td></tr><tr><td>Ethanolamine</td><td>Sigma-Aldrich</td><td>E9508</td><td></td></tr><tr><td>Neutraavidin</td><td>Thermo</td><td>31000</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Thermo</td><td>28374</td><td></td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>P2287</td><td></td></tr><tr><td>Inverted microscope</td><td>Zeiss</td><td>Axio Observer</td><td>Microscope is equipped with 40X oil immersion objective; CO&lt;sub&gt;2&lt;/sub&gt; and humidity incubation from Pecon GmbH</td></tr><tr><td>CCD camera</td><td>Hamamatsu</td><td>Orca R2</td><td>Thermoelectrically cooled (16 bit)</td></tr><tr><td>Spectrometer</td><td>Ocean Optics</td><td>QE65Pro</td><td></td></tr><tr><td>Spectrasuite</td><td>Ocean Optics</td><td>version1.4</td><td></td></tr><tr><td>c-myc peptide HyNic Tag</td><td>Solulink</td><td>SP-E003</td><td></td></tr><tr><td>monoclonal anti-c-myc antibody</td><td>Sigma-Aldrich</td><td>M4439</td><td></td></tr><tr><td>Hybridoma cell line</td><td>ATCC</td><td>CRL-1729</td><td></td></tr><tr><td>Antibiotic Antimycotic Solution (100&amp;times;)</td><td>Sigma-Aldrich</td><td>A5955</td><td></td></tr><tr><td>Serum free media RPMI 1640&amp;nbsp;</td><td>Invitrogen</td><td>&amp;nbsp;11835-030&amp;nbsp;</td><td></td></tr><tr><td>Fetal bovine serum</td><td>ATCC</td><td>30-2020</td><td></td></tr><tr><td>Rhodamine DHPE</td><td>Life Technologies</td><td>L-1392</td><td></td></tr></tbody></table>

a label-free technique for the spatio-temporal imaging of single cell secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

In a typical live cell secretion study there are multiple modes of data collection taking place. Figure 3 shows an overlay of an LSPRi image, which highlights the square arrays, and a transmitted light illuminated image which highlights the cell at lower left. Data is typically collected over a 3-hr period followed by the introduction of a saturating solution of the analyte for the normalization calculation described below. Fluorescence imagery can also be integrated into the data collection routine by the automated switching of a filter cube. In Figure 4 a cell stained with the fluorescent membrane dye rhodamine DHPE exhibits lamellipodia-like extensions (arrows). If such extensions were to overlap with the arrays they would give a false-positive for protein secretion. Having multiple modes of imagery can help identify such occurrences.
Figure 5 shows spectrometry data before and after the introduction of a saturating solution (400 nM) of commercially purchased anti-c-myc antibodies to the c-myc functionalized arrays. No cells were present in this experiment. The spectrum displays both a red shift and an increase in intensity. The difference between the areas under the two curves results in an increase in the array image intensity in LSPRi mode on the CCD camera. A non-linear least squares data analysis approach has been developed to infer fractional occupancy of surface bound ligands from the spectra.30,31
At the end of the experiment, the saturated intensity values (i.e.,&#160;fractional occupancy &#8776; 1) are used to calculate a normalized response for each array using the following formula:
Where are the normalized intensity at time point t, initial intensity at the start of the experiment, final saturated intensity, and measured intensity of the array at time point t, respectively.
Normalized values from two arrays are shown in Figure 6. One array was within 10 &#181;m of the cell under investigation while the other, used as a control, was a distance of 130 &#181;m from the cell. The sudden increase in the normalized response of the array closest to the cell relative to the flat response of the control array is indicative of a localized burst of secreted antibodies.
<img alt="Figure 1" src="/files/ftp_upload/53120/53120fig1.jpg" /> 
Figure 1. Sensor Design. A drawing depicting the geometry of a typical live cell secretion experiment. The cell (blue spheroid) is deposited on to the LSPR chip which contains arrays of biofunctionalized gold nanostructures. In the zoomed-in view, the cell secretion of interest, in this case antibodies shown as Y-shaped molecules, is measured as they bind to the surface of the functionalized nanostructures. <a href="https://www.jove.com/files/ftp_upload/53120/53120fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53120/53120fig2.jpg" /> 
Figure 2. Optical Setup. The illuminated light from a halogen lamp is first filtered by a long pass filter (LP). The light is linearly polarized (P1) and illuminates the sample via a 40X/1.4 NA objective. The scattered light is collected by the objective and passed through a crossed polarizer (P2). A 50/50 beam splitter (BS) is inserted into the collected light path for simultaneous spectroscopic and imagery analysis. Top Right: An atomic force microscopy image of 9 individual nanostructures separated by a pitch of 300 nm. <a href="https://www.jove.com/files/ftp_upload/53120/53120fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53120/53120fig3.jpg" /> 
Figure 3. Live Cell LSPRi Study. A merged transmitted light and LSPRi image showing a single hybridoma cell (lower left) surrounded by 12 arrays. This is a contrast enhanced image. Scale bar is 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/53120/53120fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53120/53120fig4.jpg" /> 
Figure 4. Live Cell Fluorescence Study. A fluorescent false color image of a single hybridoma cell stained with rhodamine DHPE, which is a membrane dye. In fluorescent imaging mode the arrays are not generally visible, however, a nearby array is observable here as a black square in the lower right corner. The cell can be seen to be separated from the array although tentacle-like extensions (possibly filopodia or lamellipodia) are extending outward from the cells (arrows). Scale bar is 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/53120/53120fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53120/53120fig5.jpg" /> 
Figure 5. Spectral Modality. The spectra obtained from a c-myc functionalized array before and after the introduction of 400 nM solution of anti-c-myc antibodies. No cells were present in this study. <a href="https://www.jove.com/files/ftp_upload/53120/53120fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/53120/53120fig6.jpg" /> 
Figure 6. Single Cell Secretion. The response of an array located within 15 &#181;m of a single cell and one located 130 &#181;m away (Control). Scale bar is 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/53120/53120fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions at JoVE.com

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

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Bioengineering

8.5K Views.  Naval Research Laboratory. Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

Video: A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and millisecond temporal resolution. These capabilities make single-molecule localization microscopy ideally suited to study molecular level biological functions in physiologically relevant environments. Here, we demonstrate an integrated protocol for both acquisition and processing/analysis of single-molecule tracking data to extract the different diffusive states a protein of interest may exhibit. This information can be used to quantify molecular complex formation in living cells. We provide a detailed description of a camera-based 3D single-molecule localization experiment, as well as the subsequent data processing steps that yield the trajectories of individual molecules. These trajectories are then analyzed using a numerical analysis framework to extract the prevalent diffusive states of the fluorescently labeled molecules and the relative abundance of these states. The analysis framework is based on stochastic simulations of intracellular Brownian diffusion trajectories that are spatially confined by an arbitrary cell geometry. Based on the simulated trajectories, raw single-molecule images are generated and analyzed in the same way as experimental images. In this way, experimental precision and accuracy limitations, which are difficult to calibrate experimentally, are explicitly incorporated into the analysis workflow. The diffusion coefficient and relative population fractions of the prevalent diffusive states are determined by fitting the distributions of experimental values using linear combinations of simulated distributions. We demonstrate the utility of our protocol by resolving the diffusive states of a protein that exhibits different diffusive states upon forming homo- and hetero-oligomeric complexes in the cytosol of a bacterial pathogen.

3D single-molecule localization microscopy is utilized to probe the spatial positions and motion trajectories of fluorescently labeled proteins in living bacterial cells. The experimental and data analysis protocol described herein determines the prevalent diffusive behaviors of cytosolic proteins based on pooled single-molecule trajectories.

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and ...

Biochemistry

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Label-Free Imaging of Single Proteins Secreted from Living Cells via iSCAT Microscopy

We demonstrate interferometric scattering (iSCAT) microscopy, a method capable of detecting single unlabeled proteins secreted from individual living cells in real time. In this protocol, we cover the fundamental steps to realize an iSCAT microscope and complement it with additional imaging channels to monitor the viability of a cell under study. Following this, we use the method for real-time detection of single proteins as they are secreted from a living cell which we demonstrate with an immortalized B-cell line (Laz388). Necessary steps concerning the preparation of microscope and sample as well as the analysis of the recorded data are discussed. The video protocol demonstrates that iSCAT microscopy offers a straightforward method to study secretion at the single-molecule level.

We present a protocol for the real-time optical detection of single unlabeled proteins as they are secreted from living cells. This is based on interferometric scattering (iSCAT) microscopy, which can be applied to a variety of different biological systems and configurations.

We demonstrate interferometric scattering (iSCAT) microscopy, a method capable of detecting single unlabeled proteins secreted from individual living ...

Engineering

Mapping the Emergent Spatial Organization of Mammalian Cells using Micropatterns and Quantitative Imaging

A fundamental goal in biology is to understand how patterns emerge during development. Several groups have shown that patterning can be achieved in vitro when stem cells are spatially confined onto micropatterns, thus setting up experimental models which offer unique opportunities to identify, in vitro, the fundamental principles of biological organisation.
Here we describe our own implementation of the methodology. We adapted a photo-patterning technique to reduce the need for specialized equipment to make it easier to establish the method in a standard cell biology laboratory. We also developed a free, open-source and easy to install image analysis framework in order to precisely measure the preferential positioning of sub-populations of cells within colonies of standard shapes and sizes. This method makes it possible to reveal the existence of patterning events even in seemingly disorganized populations of cells. The technique provides quantitative insights and can be used to decouple influences of the environment (e.g., physical cues or endogenous signaling), on a given patterning process.

The method presented here uses micropatterning together with quantitative imaging to reveal spatial organization within mammalian cultures. The technique is easy to establish in a standard cell biology laboratory and offers a tractable system to study patterning in vitro.

A fundamental goal in biology is to understand how patterns emerge during development. Several groups have shown that patterning can be achieved in vitro ...

Developmental Biology

A Microfluidic System with Surface Patterning for Investigating Cavitation Bubble(s)&#8211;Cell Interaction and the Resultant Bioeffects at the Single-cell Level

In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass substrate, that precisely controls the generation of tandem bubbles and individual cells patterned nearby with well-defined locations and shapes. We then demonstrate the generation of tandem bubbles by using two pulsed lasers illuminating a pair of gold dots with a few-microsecond time delay. We visualize the bubble-bubble interaction and jet formation by high-speed imaging and characterize the resultant flow field using particle image velocimetry (PIV). Finally, we present some applications of this technique for single cell analysis, including cell membrane poration with macromolecule uptake, localized membrane deformation determined by the displacements of attached integrin-binding beads, and intracellular calcium response from ratiometric imaging. Our results show that a fast and directional jetting flow is produced by the tandem bubble interaction, which can impose a highly localized shear stress on the surface of a cell grown in close proximity. Furthermore, different bioeffects can be induced by altering the strength of the jetting flow by adjusting the standoff distance from the cell to the tandem bubbles.

A microfluidic chip was fabricated to produce pairs of gold dots for tandem bubble generation and fibronectin-coated islands for single-cell patterning nearby. The resultant flow field was characterized by particle image velocimetry and was employed to study various bioeffects, including cell membrane poration, membrane deformation, and intracellular calcium response.

In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass ...

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell interaction known as the immunological synapse. F-actin is also speculated to play a role in regulating molecular distributions at the membrane of cells including sub-membranous vesicle dynamics and protein clustering. While standard light microscope techniques allow generalized and diffraction-limited observations to be made, many cellular and molecular events including clustering and molecular flow occur in populations at length-scales far below the resolving power of standard light microscopy. By combining total internal reflection fluorescence with the super resolution imaging method structured illumination microscopy, the two-dimensional molecular flow of F-actin at the immune synapse of T cells was recorded. Spatio-temporal image correlation spectroscopy (STICS) was then applied, which generates quantifiable results in the form of velocity histograms and vector maps representing flow directionality and magnitude. This protocol describes the combination of super-resolution imaging and STICS techniques to generate flow vectors at sub-diffraction levels of detail. This technique was used to confirm an actin flow that is symmetrically retrograde and centripetal throughout the periphery of T cells upon synapse formation.

To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell ...

Immunology and Infection

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

Biology

Quantifying Spatiotemporal Parameters of Cellular Exocytosis in Micropatterned Cells

Live imaging of the pHluorin tagged Soluble N-ethylmaleimide-sensitive-factor Attachment protein REceptor (v-SNARE) Vesicle-associated membrane protein 7 (VAMP7) by total internal reflection fluorescence microscopy (TIRFM) is a straightforward way to explore secretion from the lysosomal compartment. Taking advantage of cell culture on micropatterned surfaces to normalize cell shape, a variety of statistical tools were employed to perform a spatial analysis of secretory patterns. Using Ripley&#8217;s K function and a statistical test based on the nearest neighbor distance (NND), we confirmed that secretion from lysosomes is not a random process but shows significant clustering. Of note, our analysis revealed that exocytosis events are also clustered in nonadhesion areas, indicating that adhesion molecules are not the only structures that can induce secretory hot spots at the plasma membrane. Still, we found that cell adhesion enhances clustering. In addition to precisely defined adhesive and nonadhesive areas, the circular geometry of these micropatterns allows the use of polar coordinates, simplifying analyses. We used Kernel Density Estimation (KDE) and the cumulative distribution function on polar coordinates of exocytosis events to identify enriched areas of exocytosis. In ring-shaped micropattern cells, clustering occurred at the border between the adhesive and nonadhesive areas. Our analysis illustrates how statistical tools can be employed to investigate spatial distributions of diverse biological processes.

Live imaging of lysosomal exocytosis on micropatterned cells allows a spatial quantification of this process. Morphology normalization using micropatterns is an outstanding tool to uncover general rules about the spatial distribution of cellular processes.

Live imaging of the pHluorin tagged Soluble N-ethylmaleimide-sensitive-factor Attachment protein REceptor (v-SNARE) Vesicle-associated membrane protein 7 ...

Live-cell Imaging of Single-Cell Arrays (LISCA) - a Versatile Technique to Quantify Cellular Kinetics

Live-cell Imaging of Single-Cell Arrays (LISCA) is a versatile method to collect time courses of fluorescence signals from individual cells in high throughput. In general, the acquisition of single-cell time courses from cultured cells is hampered by cell motility and diversity of cell shapes. Adhesive micro-arrays standardize single-cell conditions and facilitate image analysis. LISCA combines single-cell microarrays with scanning time-lapse microscopy and automated image processing. Here, we describe the experimental steps of taking single-cell fluorescence time courses in a LISCA format. We transfect cells adherent to a micropatterned array using mRNA encoding for enhanced green fluorescent protein (eGFP) and monitor the eGFP expression kinetics of hundreds of cells in parallel via scanning time-lapse microscopy. The image data stacks are automatically processed by newly developed software that integrates fluorescence intensity over selected cell contours to generate single-cell fluorescence time courses. We demonstrate that eGFP expression time courses after mRNA transfection are well described by a simple kinetic translation model that reveals expression and degradation rates of mRNA. Further applications of LISCA for event time correlations of multiple markers in the context of signaling apoptosis are discussed.

Live-cell Imaging of Single-Cell Arrays LISCA - a Versatile Technique to Quantify Cellular Kinetics

We present a method for the acquisition of fluorescence reporter time courses from single cells using micropatterned arrays. The protocol describes the preparation of single-cell arrays, the setup and operation of live-cell scanning time-lapse microscopy and an open-source image analysis tool for automated preselection, visual control and tracking of cell-integrated fluorescence time courses per adhesion site.

Live-cell Imaging of Single-Cell Arrays (LISCA) is a versatile method to collect time courses of fluorescence signals from individual cells in high ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

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