Nanoplasmonic 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-10641705-B2
Publication Date
2020-05-05
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 a chip for localized surface plasmon resonance (LSPR) imaging. This chip consists of a transparent glass coverslip, compatible with standard microscopes, onto which at least one array of functionalized plasmonic nanostructures is patterned. Protein secretions from cells placed on the chip are spatially and temporally mapped by LSPR imaging, with the option to simultaneously perform transmitted light imaging and/or fluorescence imaging.
The problem addressed relates to the measurement of paracrine signaling via secreted proteins, which is critical for understanding processes such as wound healing, angiogenesis, and immune responses. Existing methods rely on fluorescent protein tags or immunosandwich assays that are limited by issues such as interference with secretion, diffuse signals in space and time, poor temporal resolution (hours to days), and the need for labeling. These shortcomings limit the ability to quantitatively track secreted proteins from individual cells at high spatio-temporal resolutions.
The invention overcomes these limitations by enabling real-time, high temporal resolution (limited primarily by camera exposure time) and label-free detection of secreted proteins. The approach benefits from Au plasmonic nanostructures lithographically patterned on standard glass coverslips allowing integration with fluorescence and bright field imaging. Quantitative measurements of secreted protein concentration are achieved as a function of time and space. The methodology applies to both adherent and non-adherent cell lines, does not suffer from photobleaching or blinking issues common to fluorescent probes, and enables detection of both continuous and burst-like secretion events.
Claims Coverage
The patent includes one independent claim describing a label-free method for real-time spatio-temporal mapping of protein secretions from individual cells using a specialized chip. This claim entails several main inventive features.
Chip with functionalized plasmonic nanostructures
A chip comprising a transparent glass coverslip compatible with standard microscopes, patterned with at least one array of functionalized plasmonic nanostructures that include a binding reagent selective for protein secretions.
Placement of cells and LSPR imaging
Placing at least one cell on the chip and spatially and temporally mapping the protein secretions from the cell using localized surface plasmon resonance (LSPR) imaging.
Simultaneous imaging modalities
Simultaneously monitoring cell secretions using transmitted light imaging, fluorescence imaging, or any combination thereof to complement LSPR imaging.
Use of gold nanostructures
Functionalization of plasmonic nanostructures with gold to serve as the sensing element of the arrays.
Proximity of cell to arrays
Ensuring that the center of the cell is no more than 15 micrometers away from at least one plasmonic nanostructure array to facilitate sensitive detection of secretions.
Normalization of array responses
Normalization of each array's response individually to account for variations and enable quantitative analysis.
Control arrays distant from cells
Utilizing at least one array as a control array located at least 65 micrometers away from the cell to distinguish localized secretions from global variations.
Overall, the inventive features cover a label-free, real-time, and spatio-temporal protein secretion mapping method using a functionalized plasmonic nanostructure chip with integrated control arrays and complementary imaging techniques.
Stated Advantages
Real-time measurement of protein secretions with temporal resolution limited mainly by camera exposure time, typically 250-400 ms.
Integration with standard glass coverslips allows simultaneous use of conventional fluorescence and bright field imaging techniques for morphological and intracellular monitoring.
Quantitative determination of secreted protein concentration as a function of time and space via calibrated nanostructures.
Incorporation of control arrays sufficiently distant from cells enables distinction between localized secretions and global signal variations.
Applicability to both adherent and non-adherent cell types.
Avoidance of photobleaching and blinking effects associated with fluorescent probes through use of Au plasmonic nanostructures.
Documented Applications
Spatial and temporal mapping of protein secretions from individual cells in real time to study paracrine signaling.
Studying extracellular signaling, including secretion bursts and continuous secretion from hybridoma cells producing anti-c-myc antibodies.
Integration with fluorescence and transmitted light imaging for simultaneous monitoring of cell morphology and intracellular markers alongside secretions.
Quantitative analysis of secretion rates, diffusion constants, and spatial distribution of secreted proteins from single cells.
Application to both adherent and non-adherent cell lines in standard microscopy and culture setups.
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