STED Microscopy Light Dose Reduction via Preliminary Scanning
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Solution Overview
Problem
High-resolution imaging of samples marked with fluorescence markers faces challenges due to light-induced bleaching and phototoxicity, particularly with high-intensity light doses in STED microscopy, which can damage living biological samples.
Innovation Solution
The method involves superimposing an intensity maximum of focused fluorescence excitation light with an intensity minimum of focused fluorescence inhibition light, selectively scanning partial areas of interest with the inhibition light, and terminating exposure based on predetermined light amounts and times to reduce the light dose and prevent bleaching.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If high-intensity fluorescence inhibition light is used in STED microscopy to achieve high-resolution imaging, then spatial resolution is improved, but phototoxicity and bleaching of fluorescence markers increase
Solution Approach 1:
The patent applies preliminary action by performing a first scan of the sample to identify areas containing fluorescence markers before performing the high-resolution STED imaging. This preliminary scanning step allows the system to pre-mark or record the locations of interest, so that subsequent high-intensity inhibition light is applied only to these specific areas rather than uniformly across the entire sample. This resolves the contradiction by enabling high spatial resolution imaging only where needed, thereby reducing overall phototoxicity and bleaching while maintaining measurement precision in the regions of interest.
2Loss of information
If the sample is scanned continuously with light intensity distribution, then imaging completeness is improved, but light dose to the sample increases causing more bleaching
Solution Approach 1:
The patent applies local quality by differentiating between different regions of the sample and applying different imaging strategies to each. Areas identified as containing fluorescence markers receive the full STED imaging protocol with high-intensity inhibition light for maximum resolution, while areas without markers receive reduced or no inhibition light exposure. This localized approach ensures imaging completeness for all regions while preserving fluorescence marker integrity in areas where high-intensity light would cause excessive bleaching.
3Measurement precision
If fluorescence inhibition light is applied to reduce the illuminated volume, then spatial resolution below diffraction barrier is achieved, but sample alteration increases
Solution Approach 1:
The patent uses preliminary scanning to identify and mark areas containing fluorescence markers before applying the fluorescence inhibition light. This allows the system to restrict the application of high-intensity inhibition light to only those specific locations where markers are present, rather than illuminating the entire sample volume. Consequently, sub-diffraction spatial resolution is achieved at marker locations while sample alteration is minimized in surrounding areas by avoiding unnecessary high-intensity light exposure.
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 significantly reduces the light dose to the sample, minimizing bleaching and phototoxicity while maintaining high-resolution imaging, especially in living biological samples.
Implementation Method 1
the sample is subjected to a light intensity distribution including an intensity maximum of focused fluorescence excitation light... fluorescence light emitted out of the sample is registered
Implementation Method 2
the fluorescence inhibition light may inhibit the fluorescence of the fluorescence markers by which the structure of interest of the sample is marked in different ways, like for example by stimulated emission in STED fluorescence microscopy
Implementation Method 3
by transferring the fluorescence markers into a dark state in which they are not able to fluoresce in RESOLFT fluorescence microscopy using switchable fluorophores
Data Source
AI summary
In methods of high-resolution imaging a structure of a sample, the structure being marked with fluorescence markers, the sample is subjected to a light intensity distribution including an intensity maximum of focused fluorescence excitation light to selectively scan partial areas of interest of the sample. Fluorescence light emitted out of the sample is registered and allocated to a respective location of the light intensity distribution in the sample. The subjection of the sample to at least one part of the light intensity distribution is terminated at each location of the light intensity distribution, if at least one criterion of the following criteria is met: (a) a predetermined maximum light amount of the fluorescence light emitted out of the sample has been registered, and (b) a predetermined minimum light amount of the fluorescence light emitted out of the sample has not been registered within a predetermined period of time.