Fluorescence scanning system for analytical ultracentrifugation
Inventors
Schuck, Peter W. • Kakareka, John W. • Pohida, Thomas J. • Patterson, George • Zhao, Hauying
Assignees
US Department of Health and Human Services
Publication Number
US-12287265-B2
Publication Date
2025-04-29
Expiration Date
2041-03-04
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Abstract
The present disclosure provides various embodiments of a fluorescence scanning system having a sample holder with a sample suspended within that is rotated by a centrifuge such that the sample is illuminated at various angles by an excitation beam by operation of a galvanometer and such that the sample emits a fluorescence emissions that is detected through a narrow window of exposure defined along the travel path of rotation taken by the sample holder when rotated by the centrifuge. A stationary fluorescence detector is in operative communication with the sample holder along the narrow window of exposure for detecting the fluorescence emissions emitted by the sample from the sample holder while also separating the excitation beam from the fluorescence emissions.
Core Innovation
The invention provides a fluorescence scanning system for analytical ultracentrifugation (AUC) that integrates a novel sample holder and scanning arrangement. The system includes a centrifuge chamber with a rotor that rotates a sample holder containing a sample suspended within. The sample is illuminated at various angles by a collimated excitation beam that is scanned by a galvanometer, enabling radial scanning of the sample. Fluorescence emissions from the sample are detected through a narrow window of exposure along the rotational travel path by a stationary fluorescence detector positioned outside the centrifuge chamber.
The system features distal and proximal plano-convex lens windows made of sapphire on opposite ends of the sample holder. These lens windows focus the excitation beam and the emitted fluorescence respectively, enabling optimal illumination and detection despite the spatial constraints inside the rotor chamber. The design also includes a stationary mirror oriented at 45 degrees relative to the galvanometer to direct the excitation beam into the sample holder, and a dichroic mirror in the detector to separate excitation and fluorescence light paths.
The problem addressed is the difficulty of detecting fluorescence emissions in analytical ultracentrifugation due to the high vacuum environment and limited space for optical elements inside the rotor chamber. Existing AUC techniques measure radial concentration distributions via interferometry and absorbance or fluorescence scanners but integrating fluorescence detection is challenging because of spatial constraints. The disclosed fluorescence scanning system simplifies integration by externalizing illumination and detection components and using galvanometer mirrors to manipulate the excitation beam angle for accurate sample scanning.
Claims Coverage
The claims define fourteen inventive features covering the fluorescence scanning system's structure and optical components.
System structure with rotating sample holder and plano-convex lens windows
A fluorescence scanning system comprising a centrifuge with a rotor for rotating a sample holder 360 degrees along a travel path, where the sample holder includes an inner chamber bounded by proximal and distal plano-convex lens windows positioned at opposite ends.
Galvanometer scanning excitation beam at different angles
A galvanometer in operative association with an illumination source to scan a collimated excitation beam at varying angles along the sample within the sample holder, enabling radial scanning.
Stationary mirror directing excitation beam into sample holder
A stationary mirror configured to transmit the excitation beam from the galvanometer through the proximal plano-convex lens window to illuminate the sample and generate fluorescence emissions.
Stationary fluorescence detector with dichroic mirror separation
A stationary fluorescence detector positioned along the travel path of the rotating sample holder, comprising a dichroic mirror operable to separate the excitation beam (deflected) from the fluorescence emissions (transmitted) for detection.
Proximal lens window focusing excitation beam at different angles
The proximal plano-convex lens window is configured to focus the collimated excitation beam at different angles along the sample.
Distal lens window focusing fluorescence emissions to detector
The distal plano-convex lens window is configured to focus fluorescence emissions emitted by the sample to the stationary fluorescence detector.
Use of sapphire lenses for lens windows
The proximal and distal plano-convex lens windows comprise sapphire lenses that form sealed windows into the sample chamber and provide appropriate optical paths.
Convex portions orienting excitation beam parallel to rotation axis
Each plano-convex lens has a convex portion allowing the excitation beam to have a more parallel orientation relative to the centrifuge rotor's axis of rotation.
Stationary mirror orientation
The stationary mirror is oriented at a 45-degree angle relative to the galvanometer.
Sample suspension in liquid within sample holder chamber
The sample is suspended in a liquid inside the inner chamber of the sample holder.
First detection lens focusing and separating beams
The stationary fluorescence detector includes a first detection lens that focuses incoming beams and operates to separate the excitation beam from fluorescence emissions.
Dichroic mirror operation for excitation and fluorescence separation
The dichroic mirror reflects the excitation beam and allows fluorescence emissions to pass through for detection.
Opposing centrifuge apertures permitting illumination and detection
The centrifuge defines opposing apertures that permit illumination of the sample by the excitation beam and detection of fluorescence emissions along a one to three degree window of the sample holder's travel path.
Short exposure time for illumination and detection
The sample is exposed for illumination and detection within a 1 microsecond period as it passes the apertures along the 360-degree travel path.
Overall, the claims cover a fluorescence scanning system employing a rotating sample holder with specialized plano-convex lens windows and a galvanometer-based excitation beam scanning mechanism, combined with stationary optical components to efficiently separate and detect fluorescence emissions in an analytical ultracentrifuge.
Stated Advantages
The use of movable galvanometer mirrors greatly simplifies the integration of a fluorescence detector into an analytical ultracentrifuge.
Fluorescence detection extends the concentration range of analytical ultracentrifugation, enabling novel applications such as characterization of ultra-high affinity protein interactions.
The design accommodates spatial constraints within the rotor chamber by externalizing illumination and detection components and optimizing lens windows, improving fluorescence emission detection.
The system allows radial scanning of the sample by modulating the excitation beam angle, with computational compensation for non-parallel beam traversal.
Documented Applications
Characterization of ultra-high affinity protein interactions, such as antibody-antigen interactions.
Study of tracer proteins in highly concentrated solutions for the characterization of protein pharmaceuticals in serum or formulation conditions.
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