Multi-focal SIM System Resolving Resolution-Speed Trade-off
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Solution Overview
Problem
Current microscopy techniques, such as confocal and structured illumination microscopy, face limitations in resolution and scanning speed, particularly when dealing with thick or highly stained samples, due to the diffraction limit and sensitivity to shot noise.
Innovation Solution
A multi-focal structured illumination microscopy system that generates a multi-focal excitation pattern using a single light beam split into multiple beams, with a scanner to create focal points on a sample, a pinhole array to block out-of-focus emissions, and a processing system to scale and sum in-focus emissions for enhanced resolution without sacrificing scanning speed.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If confocal microscopy uses a tightly closed pinhole to eliminate out-of-focus emission light, then optical resolution beyond the diffraction limit is achieved, but the signal level of emitted light is diminished to impractical levels
Solution Approach 1:
The patent divides the single light beam into multiple beams using a beam splitter, creating multiple focal points on the sample. This segmentation allows simultaneous illumination of multiple regions, increasing the total signal level while maintaining confocal resolution through the pinhole array that selectively transmits in-focus light from each focal point.
2Measurement precision
If confocal microscopy uses a tightly closed pinhole to achieve super-resolution, then resolution is improved, but alignment between excitation beam and pinhole becomes critical and misalignment reduces light signal
Solution Approach 1:
The system segments the excitation into multiple beams and uses a corresponding pinhole array where each pinhole aligns with its associated focal point. This segmented approach distributes the alignment requirement across multiple independent channels, making the system more robust to alignment errors compared to a single critical alignment point.
3Measurement precision
If structured illumination microscopy uses spatially modulated excitation with multiple raw images to achieve double the lateral resolution, then resolution is improved, but temporal resolution is sacrificed due to acquisition time
Solution Approach 1:
The patent segments the illumination into multiple focal points that simultaneously excite different regions of the sample. By capturing all focal points in a single wide-field image rather than scanning sequentially, the system achieves super-resolution without sacrificing temporal resolution, as the entire field is imaged simultaneously.
Solution Approach 2:
The system transitions from sequential spatial scanning to simultaneous multi-point illumination by adding the dimension of parallel processing. Multiple focal points are created and imaged simultaneously in the spatial domain, eliminating the time penalty associated with sequential acquisition.
4Measurement precision
If structured illumination microscopy performs optical sectioning computationally to reject out-of-focus blur, then resolution is improved, but shot noise from background fluorescence overwhelms the in-focus signal in thick or highly stained samples
Solution Approach 1:
The patent introduces a pinhole array as a physical intermediary between the sample and detector. This intermediary selectively transmits only in-focus light while blocking out-of-focus fluorescence, providing optical sectioning that is immune to shot noise from background fluorescence. The pinholes act as spatial filters that physically separate in-focus and out-of-focus signals.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The system achieves high-resolution imaging at high scanning rates with improved signal strength and resistance to shot noise, enabling the examination of thicker samples with enhanced resolution and contrast compared to conventional methods.
Implementation Method 1
a beam splitter for splitting the single light beam into a plurality of light beams forming a multi-focal pattern
Implementation Method 2
A scanner scans the plurality of light beams that forms the multi-focal pattern onto a sample such that the sample generates a plurality of fluorescent emissions
Implementation Method 3
A focusing component then defines an aperture configured to physically block out-of-focus fluorescence emissions of the plurality of fluorescent emissions resulting from each multi-focal pattern and allows through in-focus fluorescent emissions
Implementation Method 4
A scaling component scales down the plurality of in-focus fluorescent emissions resulting from each multi-focal pattern such that each of the plurality of in-focus fluorescent emissions is scaled down by a predetermined factor
Implementation Method 5
A summing component sums each of the plurality of scaled in-focus fluorescent emissions to produce a plurality of summed, scaled in-focus fluorescent emissions that form a composite image
Data Source
AI summary
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.