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.

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			<td>25mm diameter glass coverslips</td>
			<td>Bioscience Tools&#160;</td>
			<td>CSHP-No1.5-25</td>
			<td>&#160; 170&#177;5 &#181;m is optimal</td>
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			<td>Poly-methyl methacrylate</td>
			<td>Microchem</td>
			<td>PMMA 950 A4</td>
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			<td>Ethyl lactate methyl metacrylate</td>
			<td>Microchem</td>
			<td>MMA EL6</td>
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			<td>Electron beam evaporator</td>
			<td>Temescal</td>
			<td>FC-2000</td>
			<td></td>
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			<td>Electron beam lithography</td>
			<td>Raith</td>
			<td>Series 150</td>
			<td></td>
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			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>459836</td>
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			<td>Acetone</td>
			<td>Sigma-Aldrich</td>
			<td>320110</td>
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			<td>CR-7 chromium etchant</td>
			<td>Cyantek</td>
			<td>CR-7</td>
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			<td>Scanning electron microscope</td>
			<td>Zeiss</td>
			<td>Ultra 55</td>
			<td></td>
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			<td>Atomic force microscope</td>
			<td>Veeco</td>
			<td>Nanoscope III</td>
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			<td>Plasma ashing system</td>
			<td>Technics</td>
			<td>Series 85 RIE</td>
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			<td>SH-(CH2)8-EG3-OH (SPO)&#160;</td>
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			<td>TH 001-m8.n3-0.2</td>
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			<td>SH-(CH2)11-EG3-COOH (SPC)</td>
			<td>Prochimia</td>
			<td>TH 003m11n3-0.1</td>
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			<td>SH-(CH2)11-EG3-NH2 (SPN)</td>
			<td>Prochimia</td>
			<td>TH 002-m11.n3-0.2</td>
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			<td>Surface plasmon resonance system</td>
			<td>Biorad</td>
			<td>XPR36</td>
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			<td>Bare gold chip</td>
			<td>Biorad</td>
			<td>GLC chip</td>
			<td>Plasma ashed to remove the monolayer</td>
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			<td>1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide</td>
			<td>Thermo</td>
			<td>22980</td>
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			<td>N-hydroxysuccinimide (NHS)</td>
			<td>Thermo</td>
			<td>24510</td>
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			<td>Pentylamine-Biotin&#160;&#160;&#160;&#160;&#160;</td>
			<td>Thermo</td>
			<td>21345</td>
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			<td>Ethanolamine</td>
			<td>Sigma-Aldrich</td>
			<td>E9508</td>
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			<td>Neutraavidin</td>
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			<td>31000</td>
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			<td>Phosphate buffered saline</td>
			<td>Thermo</td>
			<td>28374</td>
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			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P2287</td>
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			<td>Inverted microscope</td>
			<td>Zeiss</td>
			<td>Axio Observer</td>
			<td>Microscope is equipped with 40X oil immersion objective; CO2 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>
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		<tr>
			<td>Spectrometer</td>
			<td>Ocean Optics</td>
			<td>QE65Pro</td>
			<td></td>
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			<td>Spectrasuite</td>
			<td>Ocean Optics</td>
			<td>version1.4</td>
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		<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&#215;)</td>
			<td>Sigma-Aldrich</td>
			<td>A5955</td>
			<td></td>
		</tr>
		<tr>
			<td>Serum free media RPMI 1640&#160;</td>
			<td>Invitrogen</td>
			<td>&#160;11835-030&#160;</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 ...

a-label-free-technique-for-spatio-temporal-imaging-single-cell

Research

JoVE Journal

Bioengineering

8.5K Aufrufe.  Naval Research Laboratory.  Interzellulären Kommunikation ist wichtig für die Steuerung verschiedener physiologischer Aktivitäten innerhalb und außerhalb der Zelle. Dieser Aufsatz beschreibt ein Protokoll zum Messen der räumlich-zeitliche Art der Einzelzellsekrete. Um dies zu erreichen, ist ein multidisziplinärer Ansatz verwendet, der markierungsfreien nanoplasmonische Erfassung mit Live Cell Imaging integriert. 

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

Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular functions and events1, 2. Their spatiotemporally dynamic nature has been revealed by visualization of their activity and localization in real time3. In order to gain deeper understanding of their roles in diverse cellular functions at the molecular level, the next step should be perturbation of protein activities at a precise subcellular location and timing. 
To achieve this goal, we have developed a method for light-induced, spatio-temporally controlled activation of small GTPases by combining two techniques: (1) rapamycin-induced FKBP-FRB heterodimerization and (2) a photo-caging method of rapamycin. With the use of rapamycin-mediated FKBP-FRB heterodimerization, we have developed a method for rapidly inducible activation or inactivation of small GTPases including Rac4, Cdc424, RhoA4 and Ras5, in which rapamycin induces translocation of FKBP-fused GTPases, or their activators, to the plasma membrane where FRB is anchored. For coupling with this heterodimerization system, we have also developed a photo-caging system of rapamycin analogs. A photo-caged compound is a small molecule whose activity is suppressed with a photocleavable protecting group known as a caging group. To suppress heterodimerization activity completely, we designed a caged rapamycin that is tethered to a macromolecule such that the resulting large complex cannot cross the plasma membrane, leading to virtually no background activity as a chemical dimerizer inside cells6. Figure 1 illustrates a scheme of our system. With the combination of these two systems, we locally recruited a Rac activator to the plasma membrane on a timescale of seconds and achieved light-induced Rac activation at the subcellular level6.

A method for spatio-temporal control of small GTPase activity by light is described. This method is based on rapamycin-induced FKBP-FRB heterodimerization and photo-caging systems. Optimization of light-irradiation enables the spatio-temporally controlled activation of small GTPases at the subcellular level.

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular ...

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma membrane1. To regulate neuronal excitability, the biophysical properties of ion channels are modified by signaling proteins and molecules, which often bind to the channels themselves to form a heteromeric channel complex2,3. Traditional assays examining the interaction between channels and regulatory proteins require exogenous labels that can potentially alter the protein&#39;s behavior and decrease the physiological relevance of the target, while providing little information on the time course of interactions in living cells. Optical biosensors, such as the X-BODY Biosciences BIND Scanner system, use a novel label-free technology, resonance wavelength grating (RWG) optical biosensors, to detect changes in resonant reflected light near the biosensor. This assay allows the detection of the relative change in mass within the bottom portion of living cells adherent to the biosensor surface resulting from ligand induced changes in cell adhesion and spreading, toxicity, proliferation, and changes in protein-protein interactions near the plasma membrane. RWG optical biosensors have been used to detect changes in mass near the plasma membrane of cells following activation of G protein-coupled receptors (GPCRs), receptor tyrosine kinases, and other cell surface receptors. Ligand-induced changes in ion channel-protein interactions can also be studied using this assay. In this paper, we will describe the experimental procedure used to detect the modulation of Slack-B sodium-activated potassium (KNa) channels by GPCRs.

Optical biosensor techniques can detect changes in mass near the plasma membrane in living cells and allow one to follow cellular responses in both individual cells and populations of cells. This protocol will describe detection of the modulation of potassium channels by G-protein coupled receptors in intact cells using this approach.

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma ...

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental studies on the behavior of molecules. Single molecule detection techniques commonly utilize labels such as fluorescent tags or quantum dots, however, labels are not always available, increase cost and complexity, and can perturb the events being studied. Optical resonators have emerged as a promising means to detect single molecules without the use of labels. Currently the smallest particle detected by a non-plasmonically-enhanced bare optical resonator system in solution is a 25 nm polystyrene sphere1. We have developed a technique known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that can surpass this limit and achieve label-free single molecule detection in aqueous solution2. As signal strength scales with particle volume, our work represents a &#62; 100x improvement in the signal to noise ratio (SNR) over the current state of the art. Here the procedures behind FLOWER are presented in an effort to increase its usage in the field.

We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented.

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem cell (iPS) emerged as a new method in regenerative medicine. Although this tissue regeneration is promising, contamination with unwanted cells during tissue cultures is a major concern. Moreover, there is a safety concern regarding tumorigenicity after transplantation. Therefore, there is an urgent need for eliminating specific cells without damaging other cells that need to be protected, especially in established tissue. Here, we present a method for specific cell elimination from a mixed 3D cell culture in vitro with near infrared photoimmunotherapy (NIR-PIT) without damaging non-targeted cells. This technique enables the elimination of specific cells from mixed cell cultures or tissues.

Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, yet there is an urgent need for a cell elimination method in the tissue engineering field. Here, we present a method for specific cell elimination from a mixed 3D cell culture using near infrared photoimmunotherapy (NIR-PIT).

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem ...

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific lipid component within a membrane, as in the case of phosphoinositides (PIPs), to ensure specific subcellular localization and/or activation. PIPs and cellular PIP-binding domains have been studied extensively to better understand their role in cellular physiology. We applied a pH modulation assay on supported lipid bilayers (SLBs) as a tool to study protein-PIP interactions. In these studies, pH sensitive ortho-Sulforhodamine B conjugated phosphatidylethanolamine is used to detect protein-PIP interactions. Upon binding of a protein to a PIP-containing membrane surface, the interfacial potential is modulated (i.e. change in local pH), shifting the protonation state of the probe. A case study of the successful usage of the pH modulation assay is presented by using phospholipase C delta1 Pleckstrin Homology (PLC-&#948;1 PH) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) interaction as an example. The apparent dissociation constant (Kd,app) for this interaction was 0.39 &#177; 0.05 &#181;M, similar to Kd,app values obtained by others. As previously observed, the PLC-&#948;1 PH domain is PI(4,5)P2 specific, shows weaker binding towards phosphatidylinositol 4-phosphate, and no binding to pure phosphatidylcholine SLBs. The PIP-on-a-chip assay is advantageous over traditional PIP-binding assays, including but not limited to low sample volume and no ligand/receptor labeling requirements, the ability to test high- and low-affinity membrane interactions with both small and large molecules, and improved signal to noise ratio. Accordingly, the usage of the PIP-on-a-chip approach will facilitate the elucidation of mechanisms of a wide range of membrane interactions. Furthermore, this method could potentially be used in identifying therapeutics that modulate protein&#39;s capacity to interact with membranes.

Here we present a supported lipid bilayer in the context of a microfluidic platform to study protein-phosphoinositide interactions using a label-free method based on pH modulation.

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific ...

A Guide to Build a Highly Inclined Swept Tile Microscope for Extended Field-of-view Single-molecule Imaging

Single-molecule imaging has greatly advanced our understanding of molecular mechanisms in biological studies. However, it has been challenging to obtain large field-of-view, high-contrast images in thick cells and tissues. Here, we introduce highly inclined swept tile (HIST) microscopy that overcomes this problem. A pair of cylindrical lenses was implemented to generate an elongated excitation beam that was scanned over a large imaging area via a fast galvo mirror. A 4f configuration was used to position optical components. A scientific complementary metal-oxide semiconductor camera detected the fluorescence signal and blocked the out-of-focus background with a dynamic confocal slit synchronized with the beam sweeping. We present a step-by-step instruction on building the HIST microscope with all basic components.

A detailed instruction is described on how to build a highly inclined swept tile (HIST) microscope and its usage for single-molecule imaging.

Single-molecule imaging has greatly advanced our understanding of molecular mechanisms in biological studies. However, it has been challenging to obtain ...

Implementation of Interference Reflection Microscopy for Label-free, High-speed Imaging of Microtubules

There are several methods for visualizing purified biomolecules near surfaces. Total-internal reflection fluorescence (TIRF) microscopy is a commonly used method, but has the drawback that it requires fluorescent labeling, which can interfere with the activity of the molecules. Also, photobleaching and photodamage are concerns. In the case of microtubules, we have found that images of similar quality to TIRF can be obtained using interference reflection microscopy (IRM). This suggests that IRM might be a general technique for visualizing the dynamics of large biomolecules and oligomers in vitro. In this paper, we show how a fluorescence microscope can be modified simply to obtain IRM images. IRM is easier and considerably cheaper to implement than other contrast techniques such as differential interference contrast microcopy or interferometric scattering microscopy. It is also less susceptible to surface defects and solution impurities than darkfield microscopy. Using IRM, together with the image analysis software described in this paper, the field of view and the frame rate is limited only by the camera; with a sCMOS camera and&#160;wide-field&#160;illumination&#160;microtubule length can be measured with precision up to 20 nm with a bandwidth of 10 Hz.&#160;

This protocol is a guide for implementing interference reflection microscopy on a standard fluorescence microscope for label-free, high-contrast, high-speed imaging of microtubules using in vitro surfaces assays.

There are several methods for visualizing purified biomolecules near surfaces. Total-internal reflection fluorescence (TIRF) microscopy is a commonly used ...