Multi-focal structured illumination microscopy systems and methods
Inventors
Assignees
US Department of Health and Human Services
Publication Number
US-10042149-B2
Publication Date
2018-08-07
Expiration Date
2033-02-22
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Abstract
A multi-focal selective 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 selective illumination microscopy (SIM) system for generating multi-focal patterns of a sample. The system performs focusing, scaling, and summing operations on each multi-focal pattern in a sequence that scans the entire sample to produce a high resolution composite image. This multi-focal SIM system includes components that split a single light beam into a plurality of light beams forming multi-focal patterns, scan these patterns onto a sample to excite fluorescent emissions, focus to physically block out-of-focus emissions, scale down the in-focus emissions by a predetermined factor, and sum these scaled emissions to form a composite image.
The problem addressed is that classical fluorescence microscopy is limited by the diffraction limit of light, restricting resolution. Confocal microscopy improves resolution but requires pinhole alignment and closing, which reduces signal intensity and scanning speed. Structured illumination microscopy improves resolution but sacrifices temporal resolution and is susceptible to shot noise, especially in thick or highly stained samples. There exists a need for a structured illumination microscopy system that produces multi-focal excitation patterns for each high-resolution image without sacrificing scanning speed and is resistant to shot noise.
The invention solves these issues by providing a multi-focal SIM system that produces multi-focal excitation patterns for each image, enabling high-resolution imaging at high scanning rates without significant signal loss. The system physically rejects out-of-focus light through a pinhole operation, performs a scaling operation that locally contracts fluorescent focal points while maintaining their relative distances, and sums the scaled in-focus emissions to generate a composite image. Optionally, a deconvolution operation is applied to further enhance the image resolution. These components combined provide better performance with thick samples compared to conventional SIM systems.
Claims Coverage
The patent contains one independent claim that covers a microscopy system with multi-focal pattern generation and processing, highlighting several inventive features.
Multi-focal generation and scanning of light beams
A microscopy system capable of splitting a single light beam into multiple light beams forming multiple multi-focal patterns, each with multiple focal points, and scanning these multi-focal patterns onto a sample to generate multi-focal fluorescent emissions with defined fluorescent focal points.
Processing system for removal of out-of-focus emissions
A processing system with a processor and database that removes out-of-focus fluorescent emissions from collected data for each multi-focal pattern, isolating only in-focus fluorescent emissions.
Local contraction scaling of fluorescent emissions
The processor performs a local contraction scaling operation on in-focus fluorescent emissions such that each fluorescent focal point maintains its distance relative to other points as the entire pattern is contracted, producing scaled, in-focus fluorescent emissions.
Summation to form composite image
The processor sums each scaled, in-focus fluorescent emission to produce a composite image.
The inventive features encompass producing multi-focal illumination patterns with multiple focal points, detecting and removing out-of-focus emissions, locally contracting the in-focus emissions while preserving relative positions, and summing these scaled emissions to generate a high resolution composite image.
Stated Advantages
Achieves high image resolution without sacrificing scanning speed and signal strength compared to conventional microscopy imaging systems.
Provides better performance in imaging thick samples than currently commercially available structured illumination microscopy systems.
Reduces crosstalk by maximizing distance between nearest focal points for a given point density.
Physical pinholing operation rejects out-of-focus fluorescent emissions, improving optical sectioning and image contrast.
Local contraction scaling maintains relative focal distances while improving resolution approximately two-fold laterally and axially over widefield imaging.
Enables imaging rates of about 1 Hz with resolutions down to 145 nm laterally and 400 nm axially.
Allows acquisition of three-dimensional images of samples 5-8 times thicker than conventional SIM systems.
Documented Applications
Biological imaging of antibody-labeled microtubules in human osteosarcoma (U2OS) cells embedded in fluoromount.
Imaging dual-labeled three-dimensional samples, including immuno-labeled microtubules and stained mitochondria.
Three-dimensional imaging of thicker live samples such as live, immobilized zebrafish embryos expressing GFP transgenes labeling microtubules at depths greater than 45 μm from the coverslip.
Imaging live samples such as GFP-labeled histones in live nematode embryos, obtaining four-dimensional datasets.
Interested in licensing this patent?