Non-linear Structured Illumination Microscopy for Sub-diffraction Imaging
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Solution Overview
Problem
Current microscopy techniques, such as widefield and confocal microscopy, suffer from photodamage, photobleaching, and wasteful fluorescence excitation due to the excitation of fluorescence in every plane of the specimen, while two-photon fluorescence excitation reduces these issues but still requires high intensities that can cause nonlinear photodamage.
Innovation Solution
The method involves providing spatially-patterned activation and excitation radiation to phototransformable optical labels, creating a non-linear fluorescence emission pattern with higher-order harmonics, which is then detected and shifted to generate a sub-diffraction-limited image, reducing photodamage and out-of-focus background.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If widefield or confocal microscopy is used to illuminate the entire specimen, then fluorescence excitation occurs in every plane, but this causes photodamage, photobleaching, and wasteful fluorescence excitation
Solution Approach 1:
The patent applies local quality by using spatially-modulated illumination patterns that create localized regions of high and low intensity within the specimen plane. The illumination is structured into periodic patterns (e.g., sinusoidal or lattice patterns) that selectively excite fluorescence only in specific sub-regions, thereby reducing overall photodamage while maintaining imaging capability in the focal plane
Solution Approach 2:
The patent employs periodic action through structured illumination patterns that are modulated at specific spatial frequencies. By using periodic illumination patterns and capturing multiple images at different phases, the system achieves optical sectioning and super-resolution while distributing the photodamage burden across multiple cycles, reducing peak intensity exposure
2Object-affected harmful factors
If two-photon fluorescence excitation is used to restrict excitation to regions near the focus, then photodamage is reduced, but high intensities are still required which can give rise to nonlinear mechanisms of photodamage
Solution Approach 1:
The patent applies partial action by using linear excitation with structured illumination patterns instead of full two-photon excitation. The structured patterns provide sufficient excitation in the focal plane while using lower peak intensities, achieving the desired optical sectioning effect without the harmful nonlinear photodamage associated with high-intensity two-photon excitation
3Object-affected harmful factors
If structured illumination patterns are used to reduce photodamage, then fluorescence excitation is optimized, but multiple images must be captured and processed to generate the final image
Solution Approach 1:
The patent employs feedback through computational algorithms that process the multiple structured illumination images to reconstruct the final super-resolution image. The processing pipeline includes Fourier transform-based methods that extract high-frequency information from the modulated patterns, automatically combining multiple phase-shifted images into a single high-resolution output, thereby managing the complexity through systematic computational feedback
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 minimizes photodamage and photobleaching, allows for rapid imaging, and efficiently uses fluorescence, achieving high-resolution images with reduced background noise.
Implementation Method 1
providing spatially-patterned activation radiation to a sample that includes phototransformable optical labels ("PTOLs")
Implementation Method 2
providing spatially-patterned excitation radiation to the sample, where (a) and (b) create a non-linear fluorescence emission pattern within the sample
Implementation Method 3
create a non-linear fluorescence emission pattern within the sample, the pattern including H modulation harmonics, with H>1
Data Source
AI summary
A method includes: (a) providing spatially-patterned activation radiation to a sample that includes phototransformable labels, where an optical parameter of the spatially-patterned activation radiation varies periodically in space; (b) providing spatially-patterned excitation radiation to the sample, where an optical parameter of the spatially-patterned excitation radiation varies periodically in space, where (a) and (b) create a non-linear fluorescence emission pattern within the sample, the pattern including H modulation harmonics, with H>1. The method includes (c) detecting radiation emitted from the activated and excited labels, (d) storing detected radiation data, and (e) spatially shifting one or both of the spatially-patterned excitation radiation and the spatially-patterned activation radiation with respect to the sample to spatially shift the non-linear fluorescence emission pattern within the sample, and (f) repeating (a)-(e) at least N times, with N>2. Then, a sub-diffraction-limited final image of the sample is generated based on the stored data for the N positions of the non-linear fluorescence emission pattern within the sample.