Dynamic Optical Microscopy Light Modulation
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Solution Overview
Problem
Current fluorescence microscopy techniques, particularly TIRF microscopy, face challenges such as high costs due to adapted laser sources, non-homogeneous excitation fields, variable depth of penetration, and light scattering, which limit image resolution and quality.
Innovation Solution
A method that captures and filters light emissions from a sample to selectively modulate supercritical and subcritical luminous components, enhancing image resolution by combining image zones to emphasize differences, using filters that allow certain components to pass through while reducing others, thereby improving the quality of microscopy images.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If TIRF microscopy is used to achieve high-resolution imaging, then image resolution is improved, but device cost increases due to adapted laser sources
Solution Approach 1:
The patent replaces expensive, complex laser-based TIRF microscopy equipment with a more affordable LED-based illumination system. The LED source provides sufficient excitation without requiring the adapted laser sources and complex optical components of TIRF microscopy, thereby reducing device cost while maintaining imaging capability
Solution Approach 2:
The patent changes the illumination parameters by using LED-based light sources with specific spectral characteristics instead of laser sources. This parameter change allows achieving similar imaging results with a different, more cost-effective light source that doesn't require the complex TIRF optical configuration
2Measurement precision
If TIRF microscopy is used to achieve high-resolution imaging, then image resolution is improved, but excitation field homogeneity deteriorates
Solution Approach 1:
The patent replaces the complex TIRF illumination system with a simpler LED-based epifluorescence illumination system that inherently provides more uniform excitation across the field of view, eliminating the homogeneity problems associated with TIRF's evanescent wave illumination
3Measurement precision
If TIRF microscopy is used to achieve high-resolution imaging, then image resolution is improved, but depth of penetration becomes variable
Solution Approach 1:
The patent changes the illumination mode from TIRF's evanescent wave (exponential decay profile) to epifluorescence illumination, which provides more uniform excitation depth. This parameter change makes the depth of penetration more consistent and predictable across the field of view
4Device complexity
If conventional fluorescence microscopy is used to reduce device complexity, then device cost decreases, but image resolution deteriorates
Solution Approach 1:
The patent segments the fluorescence signal into different angular components using a spatial filter. By separating and selectively detecting supercritical (high-angle) emission components that originate from the interface region, the system achieves enhanced resolution comparable to TIRF microscopy while using conventional, cost-effective equipment
Solution Approach 2:
The patent adds an angular dimension to the detection process by using a spatial filter in the back focal plane of the objective. This dimensional approach allows selective detection of light emitted at specific angles, effectively creating an angular-resolved fluorescence measurement that provides enhanced resolution without increasing device complexity
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 method achieves a significant improvement in image resolution by up to 20-25% compared to existing methods, allowing for better visualization of biological structures near the interface and deeper penetration, especially at the glass/water interface, with potential applications in studying biological processes.
Implementation Method 1
Fluorescence microscopy is a technique that takes advantage of the phenomenon of fluorescence in order to observe various compounds. Fluorescence is the property possessed by certain bodies to emit fluorescent light by themselves.
Implementation Method 2
a mask arranged in the rear focal plane of the immersion lens or a conjugate plane of said rear focal plane, so as to obscure the fluorescence emission components of the sample in the angular directions in which the angle θ is less than or equal to a critical angle θc=arcsin (nL/ns)
Implementation Method 3
an observation device comprising a full-field immersion lens, whose numerical angular aperture, ON, is greater than or equal to 1.33 and less than or equal to ns
Data Source
AI summary
Method for observing an emission of light (14, 15) from a sample (10) in a medium (11) of refractive index nL disposed against a surface (20a) of a transparent support (20) of refractive index nS, greater than nL, the emission of light comprising luminous components oriented toward the support and forming an angle θ with a direction (20b) perpendicular to the surface (20a), said components including supercritical luminous components and critical or subcritical luminous components, the method implementing an observation device (100) capable of collecting at least part of the emission of light, of applying filters (170) to the luminous signal collected; and of transforming the filtered luminous signal into an image zone of the sample (6a, 6b); the method being characterized in that:A modulation of the filtered luminous signal is carried out, in which luminous components arising from the critical or subcritical luminous components of the emission of light are allowed to pass through so as to obtain image zones (6a, 6b) of one and the same region of interest of the sample, the modulation pertaining to all or some of the luminous components of the collected luminous signal which arise from the supercritical luminous components of the emission of light; andAt least one useful image zone (6c) of the sample is produced by combining image zones (6a, 6b), the combination evidencing differences between the image zones (6a, 6b) related to the modulation.