Illumination microscopy systems and methods
Inventors
Assignees
US Department of Health and Human Services
Publication Number
US-10656403-B2
Publication Date
2020-05-19
Expiration Date
2033-02-22
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Abstract
A structured illumination microscopy (SIM) system for generating single focal point patterns of a sample is disclosed. The SIM system performs a focusing, scaling and summing operation on each single focal point that completely scan the sample to produce a high resolution composite image.
Core Innovation
The invention is a structured illumination microscopy (SIM) system designed for generating multi-focal illumination patterns to produce high resolution composite images of samples. The system uses a light source that transmits a single light beam which is split into multiple light beams forming multi-focal patterns. These multi-focal patterns illuminate the sample, causing it to emit a plurality of fluorescent emissions. The system physically removes out-of-focus fluorescent emissions through a focusing or pinholing operation, scales down the in-focus fluorescent emissions by a predetermined factor using local contraction without altering relative distances, and sums these scaled emissions to create a high resolution composite image.
The problem addressed is the limitation of prior microscopy techniques such as classical fluorescence microscopy and confocal microscopy in terms of resolution and scanning speed. Classical fluorescence microscopy is restricted by the diffraction limit, while confocal microscopy, though improving resolution, loses signal strength due to closing the pinhole and requires precise alignment. Conventional SIM offers resolution improvement but sacrifices speed and is prone to shot noise, limiting its application in thick or heavily stained samples. The existing methods either suffer from slow scanning speed or signal loss and computational noise vulnerability in creating high-resolution images.
The disclosed multi-focal SIM system solves these issues by combining multi-focal excitation patterns that allow simultaneous scanning of multiple focal points across the sample, thereby increasing scanning speed without sacrificing signal strength. It utilizes physical pinholing to reject out-of-focus light, which is more effective than computational optical sectioning prone to noise. Further, it performs a processing operation involving focusing, scaling (local contraction), and summing of the in-focus fluorescence emissions to produce composite super-resolution images with improved image contrast and resolution. This approach enables the imaging of thicker samples and faster acquisition rates than previously possible with SIM or confocal microscopes.
Claims Coverage
The patent claims cover a microscopy system comprising components and processing methods focusing on achieving high-resolution imaging through removal of out-of-focus fluorescent emissions, scaling of in-focus emissions, and summing thereof to produce composite images. There is one independent claim with multiple dependent claims specifying processing details.
Removal of out-of-focus fluorescent emissions
The system includes a processor that removes out-of-focus fluorescence for each collected fluorescent emission to leave only in-focus fluorescent emissions for further processing.
Scaling of in-focus fluorescent emissions by local contraction
The processor scales the in-focus fluorescent emissions using a local contraction operation where each scaled emission maintains the same proportional distance to others, producing scaled in-focus fluorescent emissions.
Summing scaled in-focus fluorescent emissions to produce composite image
After scaling, the processor sums the scaled in-focus fluorescent emissions to produce a composite image representing super-resolved information from the sample.
Execution of deconvolution operation
An optional processing step where the processor performs a deconvolution operation on the scaled fluorescent emissions to enhance image quality by de-blurring the composite image.
Automatic lattice detection for illumination spot localization
The system performs automatic lattice detection to determine the positions of illumination spots (focal points) using multiple vectors including lattice vectors, shift vectors, and offset vectors, to specify precise location of each focal point.
Use of lattice, shift, and offset vectors for precise localization
Two lattice vectors specify the two-dimensional displacement between neighboring illumination spots ensuring any lattice point displaced by these vectors aligns with another point; the shift vectors specify movements of illumination spots between consecutive images; and offset vectors specify absolute positions of spots closest to the center of the pattern.
The claims collectively protect the integrated system that generates multi-focal illumination patterns, physically rejects out-of-focus light, scales and sums fluorescence emissions for high-resolution composite imaging, supplemented by automated detection of illumination spot locations and optional deconvolution to enhance imaging resolution and quality.
Stated Advantages
Provides high resolution imaging at high scanning rates without significant signal loss compared to conventional microscopy techniques.
Enables imaging of thicker samples (5-8 times thicker) than conventional structured illumination microscopy systems.
Improves resolution approximately two-fold both laterally and axially over widefield imaging.
Reduces crosstalk between focal points by optimizing multi-focal pattern arrangements, improving image contrast.
Physical pinholing effectively rejects out-of-focus light, improving optical sectioning and reducing noise compared to computational methods prone to shot noise.
Combines multi-focal excitation patterns with processing steps (focusing, scaling, summing, deconvolution) for enhanced image quality and faster acquisition rates.
Documented Applications
Imaging antibody-labeled microtubules in fixed human osteosarcoma (U2OS) cells embedded in fluoromount.
Three-dimensional imaging of dual-labeled biological samples, including microtubules and mitochondria in fixed cells.
Imaging of live zebrafish embryos expressing GFP-labeled microtubules at depths greater than 45 micrometers from the coverslip surface.
Four-dimensional super-resolution imaging datasets of GFP-labeled histones in live nematode embryos.
High-speed imaging (approximately 1 Hz acquisition rate) of fluorescent biological samples with lateral resolution near 145 nm and axial resolution near 400 nm.
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