Multi-focal structured illumination microscopy systems and methods
Inventors
Assignees
US Department of Health and Human Services
Publication Number
US-9696534-B2
Publication Date
2017-07-04
Expiration Date
2033-02-22
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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 designed to generate multi-focal patterns on a sample to produce high resolution composite images. This system performs a focusing, scaling, and summing operation on each multi-focal pattern within a sequence that completely scans the sample. The key feature includes splitting a single light beam into multiple beams forming a multi-focal pattern, scanning these onto the sample, and then using a focusing component to physically block out-of-focus fluorescence emissions while allowing in-focus emissions to pass through.
The problem being solved arises from limitations in classical fluorescence microscopy and confocal microscopy in terms of resolution and signal strength. Conventional confocal microscopy achieves only 1.41 times the diffraction-limited resolution and requires precise alignment and pinhole closure, which reduces signal strength and scanning speed. Likewise, traditional SIM systems, while providing better resolution, suffer from reduced temporal resolution and computationally performed optical sectioning prone to shot noise, making them inappropriate for thick or highly stained samples. There is a need for a SIM system that produces multi-focal excitation patterns for each high-resolution image without sacrificing scanning speed or signal strength and is resilient to shot noise effects.
The multi-focal SIM system addresses these problems by generating a multi-focal excitation pattern per image, physically rejecting out-of-focus light via a pinhole array, and performing a local contraction scaling operation where in-focus fluorescent emissions are scaled down by a fixed factor while maintaining proportional distances between focal points. These scaled emissions are then summed to produce a composite high-resolution image. This novel combination enables high-resolution imaging at high scanning rates with better performance on thick samples compared to conventional SIM and confocal microscopy systems.
Claims Coverage
The patent defines one independent system claim that covers the essential inventive features of the multi-focal structured illumination microscopy system.
Multi-focal pattern generation by splitting a single light beam
The system uses a light source transmitting a single light beam and a beam splitter to split it into multiple beams forming a multi-focal pattern for illuminating the sample.
Focusing component with aperture for physical blocking of out-of-focus fluorescence
The system includes a focusing component defining an aperture or pinhole array configured to block out-of-focus fluorescent emissions and allow only in-focus fluorescent emissions to pass through.
Scaling component for local contraction of in-focus fluorescent emissions
The scaling component scales down the in-focus fluorescent emissions by a predetermined factor, preserving the relative geometric centers and proportional distances between the focal points, effectuating a local contraction.
Detector and processor arrangement for summing scaled in-focus fluorescence emissions
A detector collects the scaled in-focus fluorescent emissions and the processor sums these scaled emissions to produce a composite high-resolution image.
The claims cover a multi-focal SIM system featuring generation of multi-focal patterns via beam splitting, physical rejection of out-of-focus light by an aperture, local contraction scaling of in-focus fluorescence while preserving geometrical relations, and summing scaled emissions to yield a high-resolution composite image.
Stated Advantages
Enables high-resolution imaging without sacrificing scanning speed or signal strength compared to conventional confocal and SIM microscopy.
Improves optical sectioning physically via pinhole rejection, which is more effective and less noise prone than computational optical sectioning in traditional SIM, facilitating imaging of thicker and highly stained samples.
Achieves roughly two-fold lateral and axial resolution improvement over widefield imaging, enhancing visualization of fine structural details.
Attains high imaging rates (e.g., 1 Hz for super-resolution images) with reduced acquisition time compared to existing microscopy systems.
Documented Applications
Biological imaging of antibody-labeled microtubules in human osteosarcoma (U2OS) cells embedded in fluoromount with lateral resolution down to 145 nm and axial resolution around 400 nm.
Three-dimensional imaging of dual-labeled samples, such as microtubules and mitochondria in fixed cells, providing enhanced image contrast and resolution over widefield microscopy.
Imaging of thick live biological samples such as zebrafish embryos, capturing volumes approximately 48 μm thick with improved optical sectioning and resolution, enabling visualization of multiple cell layers.
Four-dimensional SIM datasets of GFP-labeled histones in live nematode embryos.
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