Overlay of the two images (LSPR, transmitted light) with a map of secreted antibody concentrations as generated by Finite Element  Analyses. The colored concentration scale has units of pM and the distance scale bar is 10 μm
Overlay of the two images (LSPR, transmitted light) with a map of secreted antibody concentrations as generated by Finite Element Analyses. The colored concentration scale has units of pM and the distance scale bar is 10 μm

Background: The cellular secretion of proteins as signaling molecules is known to be foundational to developmental biology, wound healing, immunology and many diseases such as cancer. While the types of proteins secreted by cells are now well cataloged, we are only just beginning to understand and appreciate how cells organize these secretions in space and time for multi-cellular functionality. State-of-the-art measurements produce just one data point every two hours with a spatial resolution of hundreds of microns: orders of magnitude coarser than what is needed to precisely map these signaling pathways. This lack of resolution has its origins in the dominant technique for detecting cell secretions, which is based upon fluorescent labeling. The introduction of these labels necessarily halts or ends the experiment and as a result, they are only introduced after a cell has been secreting for hours or days, severely limiting both spatial and temporal resolutions.

Invention: To address this roadblock, we have developed a label-free technique based upon nanoplasmonic imaging which enables the measurement of individual cell secretions with time resolutions below one second and spatial resolutions below 10 µm. This is accomplished by lithographically patterning gold plasmonic nanostructures into arrays atop standard glass coverslips. The nanostructures are functionalized for biomolecular detection using standard thiol chemistries and the detection of analyte binding is imaged by a CCD camera. As a result, the technique integrates seamlessly on to commercially available wide-field and confocal microscopes, allowing real-time transmitted light and fluorescence imaging of the cells, as well as the plasmonic imaging of secreted proteins. We anticipate this technique will be broadly applicable to the real-time characterization of both paracrine and autocrine signaling pathways with applications in immunology, developmental biology, wound healing and numerous diseases such as cancer.

A. By using the nanoplasmonic resonances, surface plasmons  can be excited with visible light using the same optical configurations used in traditional wide-field microscopy setups. B. spectroscopic signatures are manifested as an increase in the brightness of the nanostructures
A. By using the nanoplasmonic resonances, surface plasmons can be excited with visible light using the same optical configurations used in traditional wide-field microscopy setups. B. spectroscopic signatures are manifested as an increase in the brightness of the nanostructures

Advantages: Employing nanoplasmonic imaging to the study of extracellular signaling has brought with it a number of advantages over current techniques:

  1. The protein secretions are measured in real-time with the frequency of time points limited only by the exposure time of the camera, typically 250-400 ms.
  2. The gold plasmonic nanostructures are lithographically patterned onto standard glass coverslips enabling more traditional imaging techniques such as fluorescence and bright field imagery to be readily integrated into the experiments. Thus, morphological changes and intracellular fluorescent tags can be monitored simultaneously in real time.
  3. The nanostructures are calibrated for the quantitative determination of secreted protein concentration as a function of time and space.
  4. Arrays of Au nanostructures positioned sufficiently far away from the cells can be utilized as negative control arrays used to distinguish global variations in signal, e.g. instrumental drift, from localized cell secretions.
  5. The platform is designed to accommodate a wide variety of cell types relevant to wound healing, including neurons, blood cells and epithelial cells.

The combination of these advantages enabled us to measure secreted antibodies at sub-nanomolar concentrations with unparalleled spatial and temporal resolutions, while also monitoring cell health using fluorescence and transmitted light microscopy.

Applications: Because the cellular secretion of proteins plays a central role in cellular communication, the applications for this technique span biology. Proteins secretions are utilized by cells in a diverse range of fields such as immunology, wound healing, developmental biology and diseases such as cancer. Our plasmonic imaging technique is designed to integrate seamlessly with existing commercially available wide field and confocal microscopes. As such, it is meant to enable investigators to extend their current light microscopy techniques to incorporate a measurement that was previously unattainable: real time spatio-temporal mapping of protein secretions from single cells.

Licensing Status: Licensing and collaborative research and development is available to companies with commercial interest.

Lead Inventor: Marc P. Raphael

Patents: US Patent Application No. 14/039632 entitled “Calibrating Single Plasmonic Nanostructures for Quantitative Biosensing” filed on September 27, 2013 to Marc; Raphael, Christodoulides; Joseph, Byers; Jeff

Journal Articles:

  • M. P. Raphael, J. A. Christodoulides, J. B. Delehanty, J. P. Long, J. M. Byers, “Quantitative Imaging of Protein Secretions from Single Cells in Real Time”, Biophysical Journal, 105, p.602 (2013).
  • M. P. Raphael, J. A. Christodoulides, J. B. Delehanty, J. P. Long, P. E. Pehrsson, J. M. Byers, “Quantitative LSPR Imaging for Biosensing with Single Nanostructure Resolution”, currently being revised for publication at Biophysical Journal (2012).
  • M. P. Raphael, J. A. Christodoulides, S. P. Mulvaney, M. M. Miller, J. P. Long, J. M. Byers, “A new methodology for quantitative LSPR biosensing and imaging” Analytical Chemistry, 84, p.1367 (2012).

Navy Case Numbers: 102,395; 102,043; 101,529

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Photo Gallery

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Cells are cultured and passaged using standard procedures before being transferred to the chip.
Dr. Marc Raphael; Biosensors and Electronics Section; Materials and Sensors Branch; NRL Code 6363
Dr. Joseph Christodoulides; Biosensors and Electronics Section; Materials and Sensors Branch; NRL Code 6363
Dr. James Delehanty; Laboratory for Biosensors & Biomaterials; Center for Biomolecular Science & Engineering; NRL Code 6910
Dr. Joseph Christodoulides (left) and Dr. Marc Raphael (center): Biosensors and Electronics Section; Materials and Sensors Branch; NRL Code 6363. Dr. James Delehanty (right): Laboratory for Biosensors & Biomaterials; Center for Biomolecular Science & Engineering; NRL Code 6910
Dr. Joseph Christodoulides (left) and Dr. Marc Raphael (center): Biosensors and Electronics Section; Materials and Sensors Branch; NRL Code 6363. Dr. James Delehanty (right): Laboratory for Biosensors & Biomaterials; Center for Biomolecular Science & Engineering; NRL Code 6910
The nanoplasmonic arrays are fabricated onto standard 1 inch diameter, No 1.5 glass coverslips and structurally reinforced with a silicon backing ring, visible as black circle (front-center).  The chip is loaded onto a custom built perfusion apparatus for fluid exchange (back-left).  The dimensions of this assembly are similar to those of commercially available glass bottom petri dishes (back-right) in order to ensure compatibility with the majority of microscope stage inserts.
The nanoplasmonic arrays are fabricated onto standard 1 inch diameter, No 1.5 glass coverslips and structurally reinforced with a silicon backing ring, visible as black circle between the thumb and index finger.  The chip is loaded onto a custom built perfusion apparatus for fluid exchange (back-left).  The dimensions of this assembly are similar to those of commercially available glass bottom petri dishes (back-right) in order to ensure compatibility with the majority of microscope stage inserts.
The custom made perfusion chamber is made of anodized aluminum and has inlet and outlet ports of adjustable height.  The 300 uL chamber is open so that carbon dioxide and oxygen levels in solution can be adjusted by varying their gaseous concentrations in the surrounding atmosphere.  This open configuration also allows access to the chip for applications requiring micromanipulation (i.e cell manipulation, patch clamp).
A custom made perfusion chamber and circuit board for experiments requiring electrical contact to the chip.  The perfusion chamber, shown in white, has inlet and outlet ports visible to the left and right in the image.  The chamber sits atop a circuit board (green/yellow with white interconnects).  Gold contact pads on the circuit board are wire bonded to the chip allowing for localized control of the temperature (via Joule heating) and electric field.
A custom made perfusion chamber and circuit board for experiments requiring electrical contact to the chip.  The perfusion chamber, shown in white at back-right, has inlet and outlet ports.  The chamber sits atop a circuit board (green/yellow with white interconnects) shown at back-left.  Gold contact pads on the circuit board are wire bonded to the chip allowing for localized control of the temperature (via Joule heating) and electric field.   The assembled chamber is shown in the foreground.
A circuit board used for experiments requiring electrical contact to the chip.  Gold contact pads on the circuit board are wire bonded to the chip allowing for localized control of the temperature (via Joule heating) and electric field.
The nanoplasmonic chip and assembled perfusion chamber being loaded on to the stage of a Zeiss Axio Observer microscope.  The stage is enclosed in an incubation chamber (Pecon GmbH) for active control of temperature, humidity, carbon dioxide and oxygen.
The nanoplasmonic chip and assembled perfusion chamber being loaded on to the stage of a Zeiss Axio Observer microscope.  The stage is enclosed in an incubation chamber (Pecon GmbH) for active control of temperature, humidity, carbon dioxide and oxygen.
The nanoplasmonic chip and assembled perfusion chamber being loaded on to the stage of a Zeiss Axio Observer microscope.  The stage is enclosed in an incubation chamber (Pecon GmbH) for active control of temperature, humidity, carbon dioxide and oxygen.
The nanoplasmonic chip and assembled perfusion chamber being loaded on to the stage of a Zeiss Axio Observer microscope.  The stage is enclosed in an incubation chamber (Pecon GmbH) for active control of temperature, humidity, carbon dioxide and oxygen.
The nanoplasmonic chip and assembled perfusion chamber being loaded on to the stage of a Zeiss Axio Observer microscope.  The stage is enclosed in an incubation chamber (Pecon GmbH) for active control of temperature, humidity, carbon dioxide and oxygen.
The nanoplasmonic chip and assembled perfusion chamber being loaded on to the stage of a Zeiss Axio Observer microscope.  The stage is enclosed in an incubation chamber (Pecon GmbH) for active control of temperature, humidity, carbon dioxide and oxygen.
The nanoplasmonic chip and assembled perfusion chamber being loaded on to the stage of a Zeiss Axio Observer microscope.  The stage is enclosed in an incubation chamber (Pecon GmbH) for active control of temperature, humidity, carbon dioxide and oxygen.
The nanoplasmonic chip and assembled perfusion chamber being loaded on to the stage of a Zeiss Axio Observer microscope.  The stage is enclosed in an incubation chamber (Pecon GmbH) for active control of temperature, humidity, carbon dioxide and oxygen.
The nanoplasmonic chip and assembled perfusion chamber being loaded on to the stage of a Zeiss Axio Observer microscope.  The stage is enclosed in an incubation chamber (Pecon GmbH) for active control of temperature, humidity, carbon dioxide and oxygen.
Quantitative nanoplasmonic imagery analysis of single cell secretions is performed using custom Matlab software.  All other imagery (fluorescence, DIC, brightfield) can be analyzed with commercially available software (Zeiss Zen/Axiovision, MetaMorph etc).
Cells are cultured and passaged using standard procedures before being transferred to the chip.
Cells are cultured and passaged using standard procedures before being transferred to the chip.