Multi-focal structured illumination microscopy systems and methods
Inventors
Assignees
US Department of Health and Human Services
Publication Number
US-10281702-B2
Publication Date
2019-05-07
Expiration Date
2033-02-22
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Abstract
A multi-focal structured illumination microscopy (SIM) system for generating multi-focal patterns of a sample is disclosed. The multi-focal SIM system performs a focusing, scaling and summing operation on each multi-focal pattern in a sequence of multi-focal patterns that completely scan the sample to produce a high resolution composite image.
Core Innovation
The invention disclosed is a multi-focal structured illumination microscopy (SIM) system that generates multi-focal excitation patterns of a sample to produce high resolution composite images. The system involves splitting a single light beam into multiple light beams forming multi-focal patterns that scan the sample, resulting in multiple fluorescent emissions. The method includes performing focusing operations to physically block out-of-focus fluorescence and allow in-focus fluorescence through apertures, followed by scaling down the in-focus emissions by a predetermined factor and summing these scaled emissions to form a composite high resolution image.
The problem addressed stems from limitations in classical fluorescence microscopy, where resolution is restricted by the diffraction limit, and confocal microscopy techniques that, while improving resolution by closing pinholes, reduce fluorescence signal strength and require exact alignment. Current SIM systems provide resolution enhancement but sacrifice temporal resolution and are prone to shot noise, especially in thick or highly stained samples where background fluorescence overwhelms in-focus signals. These limitations restrict imaging depth, speed, and signal strength.
The disclosed multi-focal SIM system improves over prior art by providing a means to generate multi-focal excitation patterns that allow rapid scanning without sacrificing signal strength or resolution. The system physically rejects out-of-focus light via pinhole arrays instead of solely relying on computational methods, thus reducing shot noise effects and allowing imaging of thicker samples. The system also includes computerized processing steps such as automatic lattice detection, local contraction (scaling), and summation of in-focus fluorescent emissions, followed by optional deconvolution to enhance image quality beyond the diffraction limit.
Claims Coverage
The patent includes two independent claims covering microscopy systems comprising a light source, beam splitter, scanner, detector, and a processing system with key inventive features related to out-of-focus emission removal, scaling, and image composition.
Multi-focal pattern generation and scanning
A light source transmits a single beam which is split by a beam splitter into multiple light beams forming multi-focal patterns, each with multiple focal points. These multi-focal patterns are scanned onto a sample producing multi-focal fluorescent emissions corresponding to fluorescence focal points.
Processing system for out-of-focus emission removal, scaling, and summing
A processing system with a processor and database is used to remove out-of-focus fluorescent emissions from the collected data, leaving only in-focus emissions. The processor then applies a local contraction operation that scales the in-focus emissions without altering relative distances among focal points, and finally sums these scaled emissions to form a composite image.
The invention covers systems that generate multiple light beam patterns for sample illumination, physically and computationally eliminate out-of-focus light, scale the in-focus fluorescent emissions in a spatially consistent manner, and sum these to produce high resolution composite images. Optional deconvolution further improves image clarity. Lattice detection and vector calculations enable accurate spatial processing of illumination spots.
Stated Advantages
Improves spatial resolution by approximately two-fold over conventional widefield microscopy without sacrificing scanning speed or signal strength.
Physically rejects out-of-focus light using pinhole arrays, reducing shot noise and enabling imaging of thicker and highly stained samples.
Enables faster acquisition rates (e.g., about 1 Hz super-resolution imaging) than conventional image scanning microscopy.
Allows imaging of samples 5-8 times thicker than conventional SIM systems while maintaining high resolution and contrast.
Provides enhanced axial resolution approximately twice that of widefield images.
The combination of physical pinholing and computational processing improves optical sectioning similar to confocal microscopes.
Documented Applications
Imaging antibody-labeled microtubules in human osteosarcoma (U2OS) cells embedded in fluoromount with improved resolution and contrast.
Dual-labeled three-dimensional imaging of fixed cells stained for microtubules and mitochondria with enhanced axial and lateral resolution.
Three-dimensional imaging of thicker live samples including live immobilized zebrafish embryos expressing GFP-labeled microtubules at depths over 45 micrometers.
Four-dimensional SIM datasets of GFP-labeled histones in live nematode embryos.
General biological imaging with super-resolution and optical sectioning for thick and highly fluorescent samples.
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