Systems and methods for three-dimensional fluorescence polarization via multiview imaging
Inventors
Shroff, Hari • Kumar, Abhishek • Mehta, Shalin B. • La Riviere, Patrick Jean • Oldenbourg, Rudolf • Wu, Yicong • Chandler, Talon
Assignees
University of Chicago • Marine Biological Laboratory • US Department of Health and Human Services
Publication Number
US-11680903-B2
Publication Date
2023-06-20
Expiration Date
2038-05-31
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Abstract
Systems and methods for three-dimensional fluorescence polarization excitation that generates maps of positions and orientation of fluorescent molecules in three or more dimensions are disclosed.
Core Innovation
The invention provides systems and methods for three-dimensional fluorescence polarization excitation that generate maps of positions and orientations of fluorescent molecules in three or more dimensions. This is accomplished by illuminating the sample from multiple directions, ensuring excitation of fluorescent dipoles regardless of their three-dimensional orientation. The invention employs a fluorescence microscope that captures polarized fluorescence emissions from the sample using multiple objectives oriented in non-parallel directions, allowing determination of the complete three-dimensional orientation distribution of fluorophores bound to three-dimensional structures.
The invention addresses a problem in prior polarized light microscopes that illuminate and image samples from a single viewing direction, where excitation and detection are limited primarily to planes perpendicular to the illumination/viewing axis. This limitation makes it difficult or impossible to efficiently excite or detect dipoles oriented parallel to the illumination/viewing direction and thus impairs the ability to determine full molecular orientation distributions in 3D structures.
To overcome this, the invention uses multi-view imaging, including a dual-view or potentially triple-objective configuration, with polarized excitation and orthogonal or epi-detection modes. The system illuminates the sample with polarized light beams split and directed at the sample along at least two distinct, non-parallel axes and detects fluorescence emissions along these axes alternately or simultaneously. A processor analyzes fluorescence emission data from multiple detectors to compute the position and three-dimensional orientation of each excitation dipole voxel-wise, enabling reconstruction of molecular orientation distributions with improved completeness and accuracy.
Claims Coverage
The patent includes multiple independent claims covering novel configurations and methods for three-dimensional fluorescence polarization microscopy. The claims describe systems with multi-objective arrangements and methods for polarized excitation and detection to determine dipole positions and orientations.
Multi-objective fluorescence microscopy system with polarized light beam split and non-parallel illumination and detection
The system includes a light source emitting a polarized light beam which is split into first and second polarized beams directed along first and second axes that are non-parallel. First and second objective lenses illuminate the sample along these axes and detect fluorescence emissions oriented along planes non-parallel to their respective axes. A third objective oriented along a third axis at a non-parallel angle enables detection of a third fluorescence emission.
Detection of fluorescence with alternating sequence of polarized excitation and orthogonal detection
The system alternates illumination between the first and second objectives and correspondingly alternates detection to capture fluorescence emissions from dipoles oriented along different planes, enabling detection of excitation dipoles with any orientation.
Processor-based reconstruction of three-dimensional dipole orientation distributions
Detectors communicating with a processor capture images of fluorescence emissions for various polarization states and excitation directions. The processor applies algorithms to reconstruct the positions and three-dimensional orientations of excitation dipoles in the sample.
Method for determining position and orientation of excitation dipoles via multi-view polarized excitation and detection
The method involves generating and splitting a polarized laser beam, illuminating the sample through objectives oriented along non-parallel axes, detecting fluorescence emissions with objectives oriented to detect dipoles perpendicular to their axes, and processing images for various polarization states to reconstruct dipole orientations.
The independent claims collectively cover a fluorescence microscopy system and method that use multi-view, multi-axis polarized excitation combined with orthogonal or epi-detection and computational processing to determine the three-dimensional orientations and positions of fluorescent excitation dipoles with improved accuracy over single-view systems.
Stated Advantages
Ability to excite fluorescence dipoles regardless of their three-dimensional orientation, including those aligned along the axial direction of a single objective lens.
Capability to determine the complete three-dimensional orientation distribution of fluorophores bound to 3D structures, overcoming the limitations of single-view microscopes.
Use of alternating polarized excitation and orthogonal detection to collectively detect excitation dipoles of any orientation.
Computational reconstruction algorithms enable voxel-wise mapping of fluorescent dipole orientation and position.
Documented Applications
Mapping the position and three-dimensional orientation of fluorescent molecules in biological samples.
Imaging molecular assemblies, such as actin filaments labeled with fluorescent probes, to understand orientation distributions relevant to cell function and disease.
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