Systems and methods for three-dimensional fluorescence polarization via multiview imaging
Inventors
Shroff, Hari • Kumar, Abhishek • Mehta, Shalin B. • Jean-La Riviere, Patrick • Oldenbourg, Rudolf • Wu, Yicong • Chandler, Talon
Assignees
University of Chicago • Marine Biological Laboratory • US Department of Health and Human Services
Publication Number
US-11428632-B2
Publication Date
2022-08-30
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 position and orientation of fluorescent molecules in three or more dimensions. This is achieved by exciting dipoles from multiple directions, ensuring fluorescence excitation regardless of the dipole's three-dimensional orientation. A dual-view inverted selective-plane illumination microscope (diSPIM) is used to illuminate the sample and detect polarized fluorescence emissions from two different, non-parallel directions, capturing three-dimensional orientation information.
The problem addressed is that current fluorescence microscopes illuminate and image samples from a single viewing direction, limiting the ability to efficiently excite or detect dipoles parallel to the illumination/viewing direction. This incomplete orientation detection makes it difficult or impossible to determine the complete orientation distribution of fluorophores bound to three-dimensional structures, preventing comprehensive understanding of molecular orientation critical to cellular functions and disease.
The disclosed system includes multiple objectives oriented along different axes to illuminate and detect fluorescence emissions, with polarization optics for generating polarized excitation light. The system can operate in epi-detection or orthogonal detection modes, alternating illumination and detection between objectives to detect excitation dipoles regardless of their orientation. A processor uses images captured by detectors to compute three-dimensional position and orientation of excitation dipoles in each voxel of the sample. Polarization states of the excitation light can be arbitrarily modified, and three or more objectives may be used for expanded detection.
Claims Coverage
The claims include multiple independent claims covering fluorescence microscopy systems and methods with multi-axis polarized excitation and detection to determine three-dimensional dipole orientation and position.
Fluorescence microscopy system with dual objectives alternately illuminating and detecting non-parallel polarized fluorescence emissions
A system comprising a light source, polarization optics, and a beam splitter that splits polarized excitation light into two beams illuminating a sample via two objectives oriented along non-parallel axes. The objectives detect fluorescence emissions generated by the other's illumination beam, such that one objective detects fluorescence emissions oriented along a plane non-parallel to its axis. This arrangement enables detection of excitation dipoles regardless of orientation.
Inclusion of a third objective oriented along a third non-parallel axis for additional fluorescence detection
Addition of a third objective positioned at a non-parallel angle relative to the first two objectives, equipped with a detector to collect further fluorescence emissions emitted from the sample, enhancing three-dimensional orientation detection.
Processor-based computation of three-dimensional orientation distribution of excitation dipoles
Processors communicate with detectors to receive fluorescence emission images and compute the position and three-dimensional orientation of excitation dipoles in the sample voxels, generating orientation distributions bound to three-dimensional structures.
Polarization optics for varying polarization of excitation beams before sample illumination
Use of second and third polarization optics associated with each objective to modify the polarization state of the polarized excitation beams prior to illuminating the sample, enabling polarization-resolved excitation.
Method for determining position and orientation of excitation dipoles using polarized excitation from multiple non-parallel objectives and reciprocal detection
A method involving generating a polarized laser beam split into two polarized light beams illuminating a sample through two objectives oriented along non-parallel axes. Each objective detects fluorescence emissions from excitation dipoles oriented non-parallel to the other's axis. A series of images collected are processed to compute the position and orientation of excitation dipoles, optionally with arbitrary polarization state changes and reconstruction algorithms.
The independent claims collectively cover a fluorescence microscopy system and method that employ multi-directional polarized excitation and detection using multiple objectives and dedicated polarization optics to capture comprehensive three-dimensional position and orientation data of fluorescent excitation dipoles, overcoming limitations of single-view fluorescence microscopy.
Stated Advantages
Enables excitation and detection of fluorescent dipoles regardless of their three-dimensional orientation.
Generates comprehensive maps of position and orientation of fluorescent molecules in three or more dimensions.
Improves detection efficiency for dipoles oriented parallel to conventional illumination axes.
Allows reconstruction of average dipole orientation per voxel using multi-view fluorescence images.
Facilitates analysis of molecular assemblies and their orientation critical to understanding cellular functions and disease.
Documented Applications
Fluorescence microscopy imaging of actin filaments in fixed U2OS cells labeled with Alexa Fluor 488 phalloidin to determine their three-dimensional orientation.
Mapping molecular orientation in cellular structures related to processes such as directional cell migration during wound healing or metastasis.
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