Nanosplasmonic imaging technique for the spatio-temporal mapping of single cell secretions in real time
Inventors
Raphael, Marc P. • Christodoulides, Joseph A. • Byers, Jeff M. • Delehanty, James B.
Assignees
Publication Number
US-9791368-B2
Publication Date
2017-10-17
Expiration Date
2034-03-13
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Abstract
A label-free method for the spatio-temporal mapping of protein secretions from individual cells in real time by using a chip for localized surface plasmon resonance (LSPR) imaging. The chip is a glass coverslip compatible for use in a standard microscope having at least one array of functionalized plasmonic nanostructures patterned onto it. After placing a cell on the chip, the secretions from the cell are spatially and temporally mapped using LSPR imaging. Transmitted light imaging and/or fluorescence imaging may be done simultaneously with the LSPR imaging.
Core Innovation
The invention provides a label-free method for the spatio-temporal mapping of protein secretions from individual cells in real time using localized surface plasmon resonance (LSPR) imaging. This method involves a chip comprising a glass coverslip, compatible with a standard microscope, patterned with at least one array of functionalized plasmonic nanostructures. A cell placed on the chip has its secretions spatially and temporally mapped via LSPR imaging, with simultaneous transmitted light and/or fluorescence imaging optionally performed.
The problem addressed is the difficulty in measuring paracrine signaling from individual cells due to secreted proteins being highly localized and external to the cell. Existing methods using fluorescent tags hinder secretion or result in diffuse signals that are hard to quantify. Immunosandwich assays require labeling and have lower temporal resolution, limiting studies of secretion dynamics. There is a need for a label-free, high spatio-temporal resolution technique that integrates with standard microscopy and addresses these limitations.
The invention overcomes these problems by providing real-time measurement of protein secretions with time resolution limited only by camera exposure time (typically 250-400 ms). The gold plasmonic nanostructures are lithographically patterned on standard glass coverslips, allowing integration with traditional imaging such as fluorescence and bright field. Calibration enables quantitative determination of secreted protein concentration as a function of time and space. Control arrays allow distinction between global signal variations and localized secretions. The technique applies to both adherent and non-adherent cells and eliminates issues like photobleaching and blinking found in fluorescent probes.
Claims Coverage
The patent includes one independent claim presenting the core inventive method and several dependent claims elaborating on specific features.
Label-free spatio-temporal mapping of secretions using LSPR chip
A method utilizing a chip comprising a glass coverslip compatible with a standard microscope and at least one array of functionalized plasmonic nanostructures patterned on the coverslip for localized surface plasmon resonance imaging. The nanostructures are functionalized with a self-assembled monolayer comprising SH—(CH2)8-EG3-OH and SH—(CH2)11-EG3-NH2, with the SH—(CH2)11-EG3-NH2 component covalently conjugated to a c-myc peptide. The method includes placing at least one cell on the chip and spatially and temporally mapping secretions of anti-c-myc antibodies from the cell in real time using LSPR imaging.
The claims cover a method using a functionalized plasmonic nanostructure array on a microscope-compatible glass coverslip to perform real-time, label-free spatial and temporal mapping of single-cell protein secretions through localized surface plasmon resonance imaging, with specific functionalization chemistry and configurations detailed.
Stated Advantages
Real-time measurement of protein secretions with high temporal resolution limited only by camera exposure time (250-400 ms).
Lithographically patterned gold nanostructures on standard glass coverslips enable integration with traditional imaging methods such as fluorescence and bright field microscopy for simultaneous monitoring.
Calibration allows quantitative determination of secreted protein concentration as a function of space and time.
Control arrays can distinguish global variations from localized cell secretions, enhancing measurement specificity.
Applicable to both adherent and non-adherent cell lines.
Eliminates common issues in fluorescent probes such as blinking and photobleaching, improving measurement reliability.
Documented Applications
Spatio-temporal mapping of protein secretions from individual cells in real time.
Monitoring paracrine signaling involved in processes like wound healing, angiogenesis, and immune response.
Integration with standard microscopy techniques for studying cell morphology and intracellular fluorescent tags simultaneously with secretions.
Quantitative measurements of antibody secretion bursts and continuous secretions at the single-cell level.
Use with adherent and non-adherent hybridoma cell lines for antibody secretion studies.
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