Structured Plane Illumination Microscopy for Thick Sample Imaging
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Solution Overview
Problem
Current microscopy techniques face challenges in imaging thick biological samples with high resolution and accuracy due to out-of-focus background fluorescence and photobleaching, which limits the depth of field and requires high labeling densities, making it difficult to achieve isotropic resolution in three-dimensional imaging.
Innovation Solution
The use of thin light sheets, such as those created by Bessel-like beams, combined with point accumulation for imaging of nanoscale topography (PAINT) microscopy, allows for confined illumination and stochastic binding of labels, reducing background noise and enabling high-resolution imaging of thick samples with lower labeling densities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If widefield imaging floods the specimen with light to collect light from the entire specimen simultaneously, then imaging speed is improved, but out-of-focus background information increases and resolution is limited to the focal plane
Solution Approach 1:
The patent segments the illumination into multiple structured light planes that can be independently controlled and scanned through the specimen. Instead of illuminating the entire specimen at once, the light field is divided into discrete planes that are sequentially imaged, allowing high-speed capture of each plane while maintaining resolution through structured illumination patterns.
2Measurement precision
If confocal microscopy uses a pinhole to pass only light from the focal plane to the detector, then out-of-focus background information is reduced, but imaging speed decreases due to scanning requirements
Solution Approach 1:
The patent extracts the out-of-focus background information from the detection path by using structured illumination planes that selectively excite fluorophores only in specific focal planes. The detection system captures light primarily from the illuminated plane without requiring a physical pinhole, thereby maintaining high imaging speed while achieving optical sectioning.
3Measurement precision
If high labeling densities are used to achieve high resolution in thick samples, then signal intensity is improved, but background fluorescence and photobleaching increase
Solution Approach 1:
The patent applies local quality by using structured illumination that selectively excites fluorophores only in the focal plane of interest, rather than uniformly illuminating the entire specimen. This localized excitation approach maintains high resolution with lower overall labeling densities, as each plane is imaged independently with optimized illumination, reducing background fluorescence and photobleaching in non-imaged regions.
4Length of stationary object
If the depth of field is increased to image thick samples, then field of view is improved, but axial resolution decreases
Solution Approach 1:
The patent resolves the depth-of-field versus axial-resolution contradiction by adding the time dimension to the imaging process. Multiple structured light planes at different axial positions are sequentially illuminated and imaged, allowing the system to achieve both large field of view (by scanning through depth) and high axial resolution (by maintaining thin optical sections at each plane). The final three-dimensional image is constructed by combining data from all planes.
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
This approach enables high localization precision and high labeling density imaging in multiple colors for thick samples, reducing photobleaching and phototoxicity while maintaining high axial resolution, allowing for extended observations of living cells with isotropic resolution at high volumetric frame rates.
Implementation Method 1
first labels that have a binding affinity for a first structure on or within the sample and that emit light in response to excitation light
Data Source
AI summary
A sample is bathed in a solution that includes labels having a binding affinity for a structure on the sample and that emit light in response to excitation light. A sheet of excitation light having a FWHM thickness is provided to the sample. Light emitted from labels in response to the excitation light is imaged onto a detector, where the light is imaged with a detection objective having a depth of focus comparable to or greater than the FWHM thickness. The bathing of the sample and the imaging of the light emitted from the bound labels is controlled, such that the imaged light from different individual labels bound to the structure is resolved on the detector. The providing, imaging, and controlling are repeated to image, at different times, light from labels bound to the structure at different locations on or within the sample.