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-8547533-B2
Publication Date
2013-10-01
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 discloses composite probes for super resolution optical techniques using super resolution via transiently activated quenchers (STAQ). These probes comprise a donor moiety and an acceptor moiety joined by a linker. The acceptor moiety, when excited by incident radiation to an excited state, absorbs in the donor emission region such that it quenches at least a portion of the donor moiety emission. This quenching reduces the donor population and thereby enables improved resolution by modification of the point spread function and/or nanoscale chemical reactions in a target region containing the composite probe.
The method involves irradiating a selected region of a target material comprising the composite probe by producing an exciting light beam to excite the donor moiety and a quenching light beam to excite the acceptor moiety independently. The excited acceptor acts as a transient quencher for the donor emission, quenching a portion of it. The overlap of these beams produces a modified point spread function in the optical device that is narrower than would be present without quenching, yielding super-resolution imaging or nanoscale manipulation.
The problem being solved is the diffraction limit in fluorescence microscopy which restricts imaging resolution to about 200 nm for visible light. Existing super resolution microscopy methods such as STED require large laser powers and may damage biological samples, while other techniques like STORM and PALM require long periods to collect data. The invention aims to provide improved super resolution microscopy techniques that reduce spot sizes with lower power requirements and potentially faster imaging, suitable for living biological samples and other applications.
Claims Coverage
The claims include two independent claims that cover a super-resolution method using a composite probe and a method of irradiating a selected region of a target material comprising donor and acceptor molecules. The inventive features involve the composite probe structure and the method steps for excitation and quenching to achieve a modified point spread function.
Composite probe comprising a donor moiety linked to an acceptor moiety with a linker enabling transiently activated quenching
A composite probe consisting of a donor moiety linked to an acceptor moiety via a linker, where the donor produces emission when excited at a first wavelength and the acceptor, when excited separately at a second wavelength, quenches at least a portion of the donor emission by electromagnetic coupling.
Method for irradiating a selected region to produce a modified point spread function by overlapping exciting and quenching light beams
A method involving producing an exciting light beam effective to excite the donor moiety to an excited state and a quenching light beam effective to excite the acceptor moiety to an excited state such that the acceptor quenches the donor emission; directing these beams to overlap in a target region, thereby quenching a portion of donor emission and producing a narrower point spread function than without quenching.
Use of multiple donor and acceptor moieties enabling multicolor superresolution and reduced power requirements
Employing two or more donor molecules and/or two or more transiently activated quencher molecules in a single experiment to enable multicolor superresolved imaging and/or fabrication. Multiple acceptors nearby a donor reduce power needed for quenching due to increased activation probability.
Composite probe with various quenching mechanisms and linkers enabling flexibility in applications
Quenching mechanisms include absorption, proton transfer, electron transfer, spin-orbit coupling, steric decryption, and other transiently activated quenching processes. Linkers can be flexible or rigid (e.g., polyproline) with lengths typically from 15 to 35 angstroms, allowing covalent or non-covalent linking of donor and acceptor.
Integration with scanning microscopy and gating techniques for enhanced resolution
Methods for scanning multiple selected regions using the composite probe and for time-gated detection to collect optical signals, including timing, polarization, absorbance, and temporal correlations, to enhance resolution and distinguish quenched regions spatially.
The independent claims cover the composite probe composition and methods of irradiating a target region using overlapped excitation and quenching light beams to achieve super-resolution imaging or nanoscale fabrication. The inventive features include the structure and activation of the composite probes, the quenching mechanisms, use in multicolor imaging, integration with advanced microscopy scanning and detection, and production of a narrowed point spread function for improved resolution.
Stated Advantages
Multicolor superresolved imaging and fabrication can be achieved using multiple donor and transiently activated quencher molecules in a single experiment.
Use of multiple transiently activated quenchers near a donor reduces the power needed for effective quenching, improving efficiency.
The STAQ approach allows super resolution imaging at lower laser power compared to techniques such as STED, reducing potential sample damage.
Flexibility in quenching mechanisms and linker types allows optimization for various applications, potentially enhancing timing, efficiency, and compatibility with different samples.
Documented Applications
Imaging biological samples such as cells and tissues using super resolution fluorescence microscopy.
Nanolithography, including fabrication of nanometer-scale structures by photopolymerization or etching patterns in photoresist layers.
Production of photopolymerized or photocrosslinked three-dimensional gels or constructs with controlled release, degradation, and diffusivity for entrapping bioactive agents.
Nanoscale perturbation in biological molecules and cells, including initiation of local biochemical reactions and targeted release of agents at sub-diffraction limit volumes.
Fluorescence energy transfer assays with temporally gated fluorescence to monitor switching of energy transfer states.
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