Composite probes and use thereof in super resolution methods
Inventors
Knutson, Jay R. • Griffiths, Gary L.
Assignees
US Department of Health and Human Services
Publication Number
US-9273089-B2
Publication Date
2016-03-01
Expiration Date
2030-12-28
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Abstract
Composite probes for super resolution optical techniques using super resolution via transiently activated quenchers (STAQ) include a donor moiety and an acceptor moiety joined by a linker, wherein the acceptor moiety, when excited by incident radiation, is excited to a state which, for example, absorbs in the donor emission region, such that the acceptor moiety in its excited state quenches at least a portion of the donor moiety emission. Other transiently activated quenching mechanisms and moieties could accomplish the same task by reducing donor population. Also disclosed are methods for irradiating a selected region of a target material including the composite probe, wherein the composite probe enables improved resolution by point spread function modification and/or nanoscale chemical reactions.
Core Innovation
The invention relates to composite probes for super resolution optical techniques using super resolution via transiently activated quenchers (STAQ). The composite probe comprises a donor moiety and an acceptor moiety joined by a linker. The donor moiety emits when excited by a first wavelength, and the acceptor moiety, when excited separately by a second wavelength, is excited to a state where it absorbs in the donor emission region and quenches at least a portion of the donor moiety emission. This quenching modifies the point spread function to enable improved resolution.
The problem addressed is the diffraction limit in fluorescence microscopy, which restricts resolution generally to about 200 nm for visible light. Existing super resolution methods like STED require large powers that can damage biological samples, and other methods such as STORM and PALM require long data acquisition times. Thus, super resolution microscopy techniques that allow for improved ultrafine imaging of biological samples with lower power or faster acquisition are desirable.
The disclosed STAQ method provides a new super resolution technique based on point spread function engineering by a composite excitation and quenching process, where the quenching involves transient excitation of the acceptor moiety that absorbs in the donor emission region only when excited. The composite probe and corresponding irradiation methods produce a narrower point spread function than the excitation beam alone, thus enabling higher resolution imaging or nanofabrication.
Claims Coverage
The patent includes several independent claims focusing on composite probes and methods for irradiating target materials using the probes, encompassing the composition and functional properties of the probes and the irradiation techniques used.
Composite probe with donor and transiently activated acceptor moiety linked by a linker
The probe comprises a donor moiety linked to an acceptor moiety with a linker. The donor emits when excited by a first wavelength, and the acceptor, when separately excited by a second wavelength, couples electromagnetically to the donor and quenches at least a portion of the donor emission. The acceptor moiety absorbs donor emission in its excited state but not in its ground state.
Quenching mechanisms and acceptor absorption properties
Quenching is achieved by absorption of the acceptor in a spectral region of the donor, excited state proton or electron transfer, acceptor decryption of other quenching groups such as exciplexes, spin-orbit or free radical quenchers, or contact “exchange” quenchers. The acceptor absorbs donor emission effectively when excited by the second wavelength, enabling quenching.
Linker characteristics
The linker linking donor and acceptor has length 15 to 35 Å, and may be flexible or rigid. A specific example is a polyproline linker with length between 15 and 35 Å.
Probe composition with specific donor and acceptor moieties
Donor moieties include rhodamine 6G, ALEXA 564, Rhodamine 575, sulforhodamine, TAMRA, TEXAS RED, carboxy-X-rhodamine, lucifer yellow, eosin, green fluorescent protein, yellow fluorescent protein or DsRed. Acceptor moieties include IR125, IR144, IR140, HITCI, fullerene C60, oxazine 720 and various polymethine or squarylium dyes.
Excitation wavelength ranges
The donor moiety is excited to its donor excited state by incident radiation in the range 470 to 570 nm, and the acceptor moiety is excited to its acceptor excited state by incident radiation in the range 750 to 800 nm.
Dendrimeric composite probes
The composite probe can be a dendrimer comprising multiple acceptor moieties covalently bound to a single donor moiety, enhancing quenching efficiency and enabling multiple color studies.
The claims comprehensively cover composite probes having donor moieties linked to transiently activated acceptor moieties with specified spectral properties and linkers, quenching mechanisms, excitation wavelengths, and dendrimeric structures, along with methods for irradiating target materials to achieve super resolution via point spread function modification.
Stated Advantages
Multicolor superresolved imaging and fabrication can be achieved by employing multiple donor and/or transiently activated quencher molecules concurrently.
Use of multiple transiently activated quenchers at comparable distances can reduce illumination power needs proportionally, improving efficiency.
A single quenching laser wavelength can quench several donor colors, enabling multicolor superresolved video imaging.
Documented Applications
Imaging biological samples such as cells and tissues by fluorescence microscopy with improved ultrafine resolution below diffraction limit.
Nanolithography including photopolymerization and photoinitiated etching to fabricate nanoscale structures using the composite probes in photoresist layers.
Photopolymerization and photocrosslinking precursor compositions to create three-dimensional patterned gels or constructs with controlled release and degradation properties.
In situ biochemical nanoscale perturbation within cells and biomolecules, such as targeted release of agents or initiation of local biochemical reactions with sub-diffraction limited precision.
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