Multi-focal structured illumination microscopy systems and methods
Inventors
Assignees
US Department of Health and Human Services • Government of the United States of America
Publication Number
US-11086113-B2
Publication Date
2021-08-10
Expiration Date
2037-06-06
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Abstract
Various embodiments (300, 400, 500) for a multi-focal selective illumination microscopy (SIM) system for generating multi-focal patterns of a sample are disclosed. The embodiments (300, 400, 500) of the multi-focal SIM system perform 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
The invention relates to multi-focal structured illumination microscopy (MSIM) systems and methods that produce multi-focal fluorescent emissions by generating multi-focal patterns of a sample. Various embodiments of the MSIM system perform focusing, scaling, and summing operations on each generated multi-focal pattern in a sequence that completely scans the sample to produce a high resolution composite image.
The background identifies the problem that classical fluorescence microscopy is limited in resolution by the diffraction limit and that confocal microscopy improves resolution but suffers from signal loss when closing the pinhole and requires precise alignment. Existing SIM techniques provide resolution enhancement but sacrifice temporal resolution and have limitations with thick or highly stained samples due to shot noise from background fluorescence. Additionally, scanning approaches with single excitation points limit scanning speed.
The disclosed MSIM system addresses the need for a structured illumination microscopy system that produces a multi-focal excitation pattern for each high resolution image without sacrificing scanning speed and resists shot noise corruption. The system includes hardware components to generate multi-focal excitation patterns for each image to achieve high resolution at high scanning rates without significant signal loss.
Further, the MSIM system performs scaling, pinholing, and summing operations using hardware arrangements such as pinholes, mirrors, and microlens arrays, avoiding reliance on computational operations that are prone to noise and speed limitations in conventional SIM systems.
Claims Coverage
The patent includes one independent claim defining a microscopy system with specific inventive features related to multi-focal pattern generation, scanning, pinholing, scaling, rescanning, and image capture.
Multi-focal pattern generation with microlens array
The system uses a first microlens array to split a single light beam into multiple light beams that form a multi-focal pattern with multiple focal points.
Scanning of multi-focal pattern onto sample
A scanner, such as a galvanometric mirror, scans the multi-focal pattern of light beams onto the sample to generate multiple fluorescent emissions corresponding to each focal point.
Physical rejection of out-of-focus emissions by pinhole array
A pinhole array physically blocks out-of-focus fluorescent emissions from the sample and allows only in-focus emissions to pass through for improved optical sectioning.
Optical scaling for local contraction of in-focus emissions
After pinhole filtering, a first relay lens focuses in-focus emissions onto mirrors that redirect the light through a second relay lens to a second microlens array, which produces a non-inverted image with one half magnification, thereby scaling down the spatial distribution of fluorescent emissions.
Rescanning the scaled, non-inverted image and capturing with camera
The scanner rescans the scaled non-inverted image of the in-focus emissions, which is captured by a camera to produce a high resolution composite image.
4f optical configurations for image relays and scanning
First and second scan lenses are arranged in a 4f configuration to create an intermediate image plane and facilitate scanning. Further scan lenses between the second microlens array and the scanner are also arranged in 4f configuration to relay the image for rescanning.
Specific positioning of scanner and microlens arrays
The scanner is positioned at the focal point between first and second scan lenses, and the second microlens array is positioned one focal length before the expected focus to allow the 1/2 magnification optics to function correctly.
The independent claim defines a microscopy system that integrates multi-focal light beam splitting, scanning, physical pinholing, optical scaling via microlens arrays and relay optics, rescanning, and image detection to produce high resolution composite images. These inventive features jointly enable high speed, high resolution imaging with improved signal collection and optical sectioning.
Stated Advantages
Achieves high image resolution at high scanning speeds without significant signal loss compared to conventional confocal microscopy and SIM systems.
Provides better performance imaging thicker samples than commercially available SIM systems by physically rejecting out-of-focus light.
Implements pinholing, scaling, and summing steps using hardware components, reducing reliance on computational processing prone to shot noise and temporal resolution limitations.
The spinning disk MSIM embodiment reduces the number of emission optics required, simplifying alignment and potentially making the technology more accessible.
Enables imaging speeds around 1 Hz with lateral resolution down to 145 nm and axial resolution approximately 400 nm, substantially improving over widefield microscopy.
Allows for thicker three-dimensional imaging volumes (5-8 times thicker than conventional SIM), demonstrated in biological samples such as live zebrafish embryos and human cells.
Documented Applications
Biological imaging of antibody-labeled microtubules in human osteosarcoma (U2OS) cells embedded in fluoromount.
Three-dimensional imaging of dual-labeled samples combining microtubules and mitochondria in fixed cells embedded in fluoromount.
Imaging of live, immobilized zebrafish embryos expressing GFP-labeled microtubules at depths greater than 45 micrometers from the coverslip surface.
Four-dimensional SIM datasets of GFP-labeled histones in live nematode embryos.
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