Multicolor Optical Scanning With Shared STED De-Excitation
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Solution Overview
Problem
Existing high-resolution optical scanning methods, such as STED microscopy, are limited by the need for monochromatic operation, which restricts the use of multiple fluorescent dyes and hinders multicolor imaging, and the high cost of de-excitation light sources due to their high power requirements.
Innovation Solution
The method and device utilize substances with distinct excitation spectra but overlapping de-excitation spectra, allowing separate excitation and de-excitation of each substance, using shared de-excitation sources and detectors, and synchronized detection to minimize interference.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If monochromatic operation is used in STED microscopy to achieve high-resolution imaging, then spatial resolution beyond the diffraction limit is achieved, but the ability to perform multicolor imaging with multiple fluorescent dyes is restricted
Solution Approach 1:
The patent segments the imaging process by wavelength channels, performing excitation and de-excitation steps sequentially for different fluorescent dyes at different wavelengths. This allows multicolor imaging while maintaining the high-resolution STED effect in each channel separately, thus resolving the contradiction between single-wavelength resolution and multicolor versatility
Solution Approach 2:
The patent employs periodic alternation between excitation and de-excitation light pulses at different wavelengths in a time-multiplexed manner. Each fluorescent dye is excited and de-excited in periodic cycles at its specific wavelength range, enabling multicolor imaging while preserving the saturated de-excitation effect for super-resolution in each periodic cycle
2Reliability
If high power de-excitation light sources are used to achieve saturated de-excitation at the focus edge, then complete de-excitation is achieved, but the cost of the equipment increases significantly
Solution Approach 1:
The patent applies de-excitation light selectively only at the focus edge region where it is most needed, rather than uniformly across the entire focal volume. This partial action approach achieves the necessary saturated de-excitation for super-resolution while using significantly less total light power, thereby reducing equipment cost and avoiding excessive photodamage
Solution Approach 2:
The patent creates a spatially non-uniform de-excitation intensity distribution where the de-excitation light intensity is highest at the focus edge and decreases toward the center. This local quality variation ensures complete de-excitation where required for resolution while minimizing overall power consumption and equipment requirements
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-resolution multicolor imaging with reduced equipment costs and improved information yield by avoiding bleaching and interference between substances.
Implementation Method 1
the sample is illuminated with focused light of a wavelength of the excitation spectrum of the substance to generate the first state Z1 of the substance
Implementation Method 2
the sample is illuminated in a focus edge region of the excitation with light of a suitable de-excitation wavelength to generate the second state Z2 of the substance
Implementation Method 3
emission light emanating from the sample, resulting from a decay of remaining first states Z1, is detected by means of a detection device
Data Source
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AI summary
The invention relates to a method for high-resolution optical scanning of a sample, preferably using a laser scanning fluorescence microscope, wherein the sample (10) comprises a substance (S1) that can be translocated into different energy states - first state Z1 and second state Z2 - wherein the first state Z1 and the second state Z2 differ from each other in at least one optical property, wherein the sample (10) is illuminated with light of a wavelength of the excitation spectrum (1) of the substance (S1) to generate the first state Z1 of the substance (S1) in a region, wherein the sample (10) is illuminated with light of a suitable de-excitation wavelength in a focus edge region of the excitation to generate the second state Z2 of the substance (S1), and wherein emission light (85) emanating from the sample (10), resulting from a decay of remaining first states Z1, is detected by means of a detection device (89).The sample (10) comprises at least one further substance (S2) that can be converted into a first state Z1' and a second state Z2', wherein the substances (S1, S2) differ from each other in at least one property and at least partially agree in at least one other property. When scanning the sample (10), the excitation and/or de-excitation and/or detection steps are performed separately for each substance (S1, S2). A corresponding apparatus is also described.