Multi-focal structured illumination microscopy systems and methods
Inventors
Assignees
US Department of Health and Human Services
Publication Number
US-10025082-B2
Publication Date
2018-07-17
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
Classical fluorescence microscopy is limited by the diffraction limit, restricting lateral resolution to about 200 nm and axial resolution to about 500 nm, which limits detailed imaging of samples. Confocal microscopy improves resolution by physically rejecting out-of-focus light using point illumination and pinholes but suffers from signal loss and requires precise alignment, making super-resolution imaging impractical at faster speeds. Structured illumination microscopy (SIM) provides double the lateral resolution but sacrifices temporal resolution because multiple raw images are required for each super-resolution image, and its computational optical-sectioning is prone to shot noise, limiting its effectiveness on thick or highly stained samples.
The invention 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 is resistant to shot noise that corrupts SIM images. The multi-focal structured illumination microscopy (MSIM) systems described include hardware components that generate multi-focal excitation patterns and perform focusing, scaling, and summing steps. This arrangement allows for high image resolution at high scanning speeds with better performance on thick samples than conventional SIM and confocal microscopy systems. The in-focus fluorescent emissions generated by the sample in response to the multi-focal patterns are physically filtered using pinholes to reject out-of-focus light, then locally contracted (scaled down) and summed to produce high-resolution composite images.
Embodiments include configurations where a single light beam is split by microlens arrays or digital micromirror devices to create multi-focal patterns with an array of focal points, which are scanned across the sample to induce fluorescence. The emitted fluorescence is de-scanned and physically filtered to isolate in-focus light, then scaled down by a predetermined factor that maintains relative spacing between focal points, and summed to form the final composite high-resolution image. The system further uses deconvolution techniques to enhance image clarity. Notably, some embodiments perform scaling, pinholing, and summing hardware operations, avoiding reliance solely on computational processing, which allows for faster image acquisition with strong signal strength.
Claims Coverage
The patent contains one independent claim describing a microscopy system with several key inventive features related to multi-focal structured illumination microscopy.
Use of microlens arrays to form multi-focal patterns
The system employs a first microlens array to split a single light beam into multiple light beams forming at least one multi-focal pattern with multiple focal points.
Scanning and rescanning of multi-focal patterns
A scanner is used to scan the plurality of light beams forming the multi-focal pattern onto a sample and to rescan the non-inverted image of these beams after passing through downstream components, ensuring precise imaging.
Physical rejection of out-of-focus fluorescent emissions
A pinhole array is utilized to block out-of-focus fluorescent emissions for each multi-focal pattern, allowing only in-focus emissions to pass through, thereby improving image contrast and optical sectioning.
Scaling and image formation through a second microlens array
A second microlens array produces a non-inverted image of the plurality of light beams at one half magnification, facilitating local contraction (scaling) of the multi-focal fluorescent emissions to achieve resolution enhancement.
Capture of the scanned non-inverted image by a camera
A camera captures the rescanned, scaled, and optically sectioned multi-focal images to produce a composite high-resolution image.
The independent claim covers a microscopy system combining microlens arrays, scanning apparatus, pinhole arrays for physical out-of-focus emission rejection, scaling via microlens optical arrangement, rescanning of the scaled image, and image capture by a camera. These inventive features cooperate to produce high-resolution, optically sectioned composite images with improved speed and signal strength compared to conventional microscopy.
Stated Advantages
Provides high image resolution at high scanning speeds without significant signal loss compared to conventional confocal and SIM systems.
Physically rejects out-of-focus fluorescence emissions using hardware components such as pinholes and microlens arrays, improving optical sectioning and image contrast in thicker samples.
Reduces shot noise impacts inherent in computational optical-sectioning of conventional SIM, enabling imaging of thicker or highly stained samples.
Maintains relative distances between focal points during scaling, preserving spatial accuracy while enhancing resolution.
Hardware-based scaling, pinholing, and summing operations reduce reliance on computational processing, enabling faster image acquisition.
Enables acquisition of super-resolution images at rates of approximately 1 Hz with lateral resolution down to about 145 nm and axial resolution about 400 nm.
Compatible with both biological and thick live samples, providing improved imaging volumes 5-8 times thicker than conventional SIM systems.
Documented Applications
Imaging antibody-labeled microtubules in human osteosarcoma (U2OS) cells embedded in fluoromount with improved resolution and contrast compared to widefield imaging.
Three-dimensional imaging of dual-labeled samples with microtubules and mitochondria, enhancing image contrast and axial resolution over widefield microscopy.
High-resolution volumetric imaging in live, immobilized zebrafish embryos expressing GFP-labeled microtubules, capturing multiple cell layers up to 48.2 microns thick.
Four-dimensional super-resolution imaging of GFP-labeled histones in live nematode embryos.
General biological microscopy requiring super-resolution imaging with high scanning speed and effective optical sectioning, suitable for thick and highly stained specimens.
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