Multi-focal structured illumination microscopy systems and methods
Inventors
Assignees
US Department of Health and Human Services
Publication Number
US-10156711-B2
Publication Date
2018-12-18
Expiration Date
2033-02-22
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Abstract
Various embodiments for a multi-focal selective illumination microscopy (SIM) system for generating multi-focal patterns of a sample are disclosed. The multi-focal SIM system performs a focusing, scaling and summing operation on each generated multi-focal pattern in a sequence of multi-focal patterns that completely scan the sample to produce a high resolution composite image.
Core Innovation
Various embodiments for a multi-focal selective illumination microscopy (SIM) system are disclosed that generate multi-focal patterns of a sample, perform focusing, scaling, and summing operations on the generated multi-focal patterns, and produce a high resolution composite image of the sample.
Classical fluorescence microscopy is limited in resolution by the diffraction limit, and while confocal microscopy improves resolution using point illumination and a pinhole arrangement, this approach reduces signal level and demands precise alignment. Structured illumination microscopy (SIM) improves lateral resolution by illuminating a sample with spatially modulated excitation intensity, but at the cost of temporal resolution and susceptibility to shot noise, making it less effective for thick or highly stained samples.
The disclosed multi-focal SIM (MSIM) system addresses these issues by generating a multi-focal excitation pattern for each image of the sample, allowing high resolution imaging at high scanning rates without significant loss of signal strength. The MSIM system physically blocks out-of-focus fluorescence emissions, scales down the in-focus emissions by a predetermined factor, and sums the scaled emissions to form a composite image. This approach improves speed, resolution, and noise resistance compared to previous microscopy techniques.
Claims Coverage
The patent includes one independent claim defining a microscopy system with multiple coordinated spinning disks for multi-focal illumination and signal processing. There are five inventive features detailing the components and their coordinated functions.
A microscopy system with coordinated spinning disks for multi-focal pattern generation and detection
The system comprises a light source transmitting a single light beam, a first spinning disk with converging microlenses rotating in a first direction to split the beam into multiple light beams forming a multi-focal pattern, a second spinning disk with a pinhole array rotating in the same direction to block out-of-focus light beams and allow in-focus light beams to pass, and a third spinning disk with diverging microlenses also rotating in the first direction, all along an optical axis and rotating in sync.
Diverging microlens focal length configured relative to converging microlenses
The third spinning disk with diverging microlenses has a focal length that is half that of the first spinning disk with converging microlenses, forming part of the optical configuration.
Equal number of microlenses on diverging and converging spinning disks
The spinning disk with diverging microlenses has the same number of microlenses as the spinning disk with converging microlenses, ensuring corresponding spatial configurations.
Spatial alignment of microlenses across spinning disks
The microlenses on the spinning disk with diverging microlenses are spaced at the same spatial locations as those on the spinning disk with converging microlenses to maintain alignment in the system.
Galilean telescope arrangement formed by spaced spinning disks
The three spinning disks are spaced apart by the difference of their focal lengths such that a Galilean telescope with magnification of one half is arranged, facilitating the desired optical processing and imaging.
The claims collectively disclose a microscopy system utilizing synchronized spinning disks with converging and diverging microlenses and a pinhole array to physically block out-of-focus light, scale in-focus emissions, and form composite images, arranged in a precise Galilean telescope configuration to achieve multi-focal structured illumination microscopy with improved resolution and imaging capability.
Stated Advantages
Produces high resolution images at high scanning speeds without significant loss in signal strength compared to conventional confocal microscopy and SIM systems.
Provides better performance in thick samples than commercially available SIM systems by physically rejecting out-of-focus light, enhancing optical sectioning.
Uses an arrangement of hardware components such as pinholes, mirrors, and microlens arrays to perform pinholing, scaling, and summing operations, reducing reliance on computational processing.
The spinning disk arrangement reduces the number of emission optics and offers easier optical alignment, facilitating practical implementation of the technology.
Enables imaging rates of 1 Hz at resolutions down to 145 nm laterally and 400 nm axially, with three-dimensional imaging 5-8 times thicker than conventional SIM.
Documented Applications
Imaging antibody-labeled microtubules in human osteosarcoma (U2OS) cells embedded in fluoromount to achieve improved resolution and contrast.
Dual-labeled, three-dimensional imaging of fixed cells with labeled microtubules and stained mitochondria, enhancing image contrast and resolution.
Three-dimensional imaging of thicker live samples such as live immobilized zebrafish embryos expressing GFP-labeled microtubules, producing volumetric images up to about 48 μm thickness.
Four-dimensional SIM datasets of GFP-labeled histones in live nematode embryos, demonstrating application to live biological samples with temporal resolution.
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