Spectroscopic measurements and super-resolution imaging by supracence
Inventors
Li, Alexander Dequan • Wan, Wei
Assignees
Washington State University WSU
Publication Number
US-11435287-B2
Publication Date
2022-09-06
Expiration Date
2039-10-17
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Abstract
Application of a new light-molecule interaction in which molecules enable emission of photons with more energy than that of the absorbed photons achieves higher resolution than fluorescence imaging. This emission phenomenon is termed supracence and is applied to obtain more information about the structure and properties of a specimen than currently possible with fluorescence imaging techniques. Because supracence originates from chemical bonds, any structure that contains chemical bonds meets the necessary condition to potentially emit supracence. Super spectral resolution images are achieved by selectively exciting a target molecule to suprace without exciting another fluorophore that has absorption and emission rather close to the target.
Core Innovation
The invention introduces a new light-molecule interaction termed supracence, in which molecules can emit photons with more energy than that of the absorbed photons. This phenomenon, unlike fluorescence where emission is of lower energy, leverages chemical bond dynamics to make possible higher energy emission. Both the excitation and emission involved in supracence are highly specific to narrow regions of the spectrum, offering the ability to selectively excite target molecules while avoiding the excitation of other fluorophores with nearby absorption and emission peaks.
The invention addresses longstanding limitations in fluorescence imaging stemming from broad and featureless emission bands, which hinder efficient wavelength multiplexing and can create significant spectral crosstalk between different probes. Fluorescence results in thermal loading of samples due to inefficient energy transitions. By contrast, supracence enables sharper spectral discrimination and minimal crosstalk, offering enhanced information about specimen structure and molecular properties, and allowing for high-resolution spectral and spatial imaging.
Through demonstrated designs of microscopes and spectrometers, supracence is shown to provide superior spectral and spatial resolution compared to existing fluorescence techniques. Experimental evidence supports that supracence is a one-photon, linear optical process drawing extra energy from molecular potential, resulting in ultra-sharp emission peaks. This specificity enables more efficient wavelength multiplexing and the accurate identification and imaging of molecular species within complex samples, surpassing the capabilities of both traditional fluorescence and Raman spectroscopies.
Claims Coverage
The patent includes five independent inventive features covering supracence-based spectroscopic and imaging methods and associated instruments.
Absorption-and-emission instrumental method for capturing supracence
A method comprising: - Directing a light beam at a sample with an excitation wavelength (λEX) to excite the sample from the lowest electronic and vibrational state to an excited state in a single step. - Filtering spectra with imaging optics to separate wavelengths less than λEX from those greater than λEX. - Capturing the filtered spectra with wavelengths less than λEX using a detector to produce output showing only supracence.
Microscope for supracence imaging
A microscope including: - At least one light source (excitation wavelength λEX) and beam-forming optics to excite a sample from its lowest electronic and vibrational state to an excited state in a single step. - Imaging optics to filter spectra so that wavelengths less than λEX are separated from those greater than λEX. - At least one detector (or distinct region) capturing only the filtered spectra with wavelengths less than λEX.
Method of imaging with minimized photo-toxicity
A method comprising: - Selecting a fluorophore with non-zero supracence for a given excitation wavelength (λEX). - Dying a sample with the selected fluorophore. - Imaging using only a laser at λEX and performing simultaneous imaging of both fluorescence and supracence.
Spectrometer for supracence measurement
A spectrometer including: - At least one light source and first optics for selecting excitation wavelength (λEX) to expose a sample and excite it from the lowest electronic and vibrational state to an excited state in a single step. - Second optics to select a single supracence spectrum (wavelengths < λEX). - One or more detectors configured to capture only the selected single supracence spectrum.
Flow cytometer for supracence-based multiplexing
A flow cytometer comprising: - n lasers with respective excitation wavelengths (λEX1 to λEXn), each optimized for a particular color. - Beam-forming optics to align laser focus spots on a single file flow cell line for excitation from the lowest electronic and vibrational state to an excited state in a single step. - For each excitation wavelength, imaging optics to filter spectra to separate wavelengths less than and greater than the excitation wavelength, and one or more detectors capturing only the filtered spectra with wavelengths less than the excitation wavelength.
These inventive features collectively define the new absorption and emission method (supracence), the associated instrument designs (microscopes, spectrometers, flow cytometers), and novel methods for selective and minimally invasive imaging, providing enhanced spectral and spatial resolution in molecular analyses.
Stated Advantages
Supracence enables superior spectral and spatial resolution compared to fluorescence imaging, allowing for more efficient wavelength multiplexing and sharper discrimination of molecular species.
The method produces minimal crosstalk between different probes, enabling clear identification and imaging even when spectral peaks are closely spaced.
Instruments and methods using supracence can measure more colors (up to ten without measurable crosstalk), significantly surpassing the limitations of fluorescence.
Simultaneous imaging of both supracence and fluorescence enables energy-neutral imaging, which minimizes photo-toxicity and preserves physiological conditions in living cells.
Supracence enables unique identification of each compound in a mixture through characteristic single narrow band emission, simplifying analysis without the need for molecular separation.
Fiber-based supracence spectrometers allow targeted diagnosis and analysis in locations inaccessible to conventional instruments, such as inside the human body or on production lines.
Supracence-based flow cytometry supports the detection of many more labeled cell phenotypes with low spectral overlap, improving analysis accuracy relative to fluorescence-based techniques.
Documented Applications
Super-resolution imaging and molecular identification in biological specimens, including distinguishing between mitochondria and endosomal/lysosomal compartments in live cells.
High-capacity multiplexed spectral imaging and analysis, resolving more molecular probes in research or diagnostic samples than possible with conventional fluorescence.
Pharmaceutical, clinical diagnosis, biotechnology, chemical, environmental, agricultural, semiconductor, and food & beverage industries for compound identification and analysis.
Non-invasive disease diagnosis, such as tumor identification in the human body or plant virus infection monitoring.
Flow cytometry for complex cell phenotype characterization, enabling more markers to be simultaneously detected with reduced crosstalk.
Fiber-based supracence spectrometry for in-situ analysis and quality control in various industrial and healthcare settings.
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