Tilted Structured Illumination Microscopy Axial Resolution
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Solution Overview
Problem
Current fluorescence microscopy techniques, such as epifluorescence microscopy, suffer from anisotropic resolution, lacking optical sectioning and experiencing the 'missing cone' phenomenon, which limits axial resolution and throughput.
Innovation Solution
The implementation of tilted structured illumination microscopy (SIM) using a tilted structured illumination pattern asymmetrical about the system's pupil center, defined by two wave vectors, to enhance axial resolution and add optical sectioning with minimal throughput degradation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If standard epifluorescence microscopy is used, then the system is simple and throughput is high, but axial resolution is poor and optical sectioning is missing
Solution Approach 1:
The patent employs an asymmetrical illumination pattern in the tilted SIM technique, where the structured illumination is deliberately tilted at an angle relative to the optical axis rather than being symmetrical. This asymmetry allows the illumination pattern to define spatial frequencies along the optical axis, thereby improving axial resolution while maintaining reasonable throughput compared to conventional symmetrical SIM approaches
Solution Approach 2:
The patent introduces tilted structured illumination that adds a dimensional component along the optical axis by tilting the illumination pattern. This approach defines spatial frequencies in the axial direction, effectively utilizing another dimension (the tilt angle) to improve axial resolution without requiring complex 3D scanning architectures
2Measurement precision
If conventional SIM techniques are used to improve axial resolution, then axial resolution increases, but throughput decreases significantly (30-fold)
Solution Approach 1:
The tilted SIM technique uses an asymmetrical, tilted illumination pattern that is not symmetrical about the pupil center, which allows achieving axial resolution improvement with fewer illumination patterns compared to conventional SIM, thereby reducing the throughput penalty from 30-fold to six-fold
Solution Approach 2:
The patent changes the parameters of the illumination pattern by introducing a tilt angle and using exactly two wave vectors with specific spatial frequencies along the optical axis. This parameter optimization allows achieving axial resolution improvement with minimal degradation to throughput
3Productivity
If ApoTome or OS-SIM techniques are used, then optical sectioning is added with minimal throughput decrease, but axial resolution is not improved
Solution Approach 1:
The tilted SIM technique uses an asymmetrical illumination pattern that defines spatial frequencies along the optical axis, simultaneously achieving both optical sectioning and doubled axial resolution, unlike ApoTome or OS-SIM which only provide optical sectioning without axial resolution improvement
Solution Approach 2:
The tilted SIM approach serves multiple functions simultaneously: it provides optical sectioning, doubles axial resolution, and maintains relatively high throughput (only six-fold decrease). This multi-functionality makes it superior to ApoTome or OS-SIM which only provide optical sectioning
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 doubles axial resolution while maintaining improved lateral resolution and optical sectioning, achieving a six-fold decrease in throughput compared to standard microscopy, outperforming existing methods by providing superior axial resolution and optical sectioning efficiency.
Implementation Method 1
fluorophores are excited by excitation light of a fluorophore-dependent excitation wavelength and then emit fluorescence emission light of a fluorophore-dependent emission wavelength
Implementation Method 2
the tilted structured illumination pattern is based on exactly two wave vectors and defines spatial frequencies along an optical axis of an objective of the system
Data Source
AI summary
A tilted structured illumination pattern for use in an optical system is generated, wherein the tilted structured illumination pattern is not symmetrical about a center point of a back pupil of the system, and wherein the tilted structured illumination pattern is based on exactly two wave vectors and defines spatial frequencies along an optical axis of an objective of the system. The tilted structured illumination pattern is directed to be incident on a sample, such that one or more fluorophores of the sample are excited by the structured illumination pattern. A phase of the tilted structured illumination pattern is varied. A set of images of fluorescence emission emitted by the sample is captured, wherein each image of the set of images corresponds to a respective phase of the tilted structured illumination pattern and to a respective axial imaging plane. Combined image data is generated based on the set of images.