STED Microscopy Adaptive Illumination for Photobleaching Reduction
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Solution Overview
Problem
Conventional STED microscopy suffers from photobleaching and high radiation exposure, limiting the ability to image samples multiple times and resulting in long measurement times due to high light intensities required for achieving increased spatial resolution, which is particularly problematic in three-dimensional imaging.
Innovation Solution
A method that generates an overview image with a lower spatial resolution before the STED imaging process to identify areas without relevant image information, allowing for reduced radiation power in those areas and adaptive modulation of the STED light intensity, enabling faster and gentler imaging by reducing unnecessary light exposure and leveraging image processing to enhance resolution.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If high light intensity is used to achieve increased spatial resolution in STED microscopy, then spatial resolution is improved, but photobleaching and radiation exposure increase
Solution Approach 1:
The patent applies preliminary action by generating an overview image at lower resolution before the main STED imaging process. This overview image is used to identify regions of interest, allowing the system to pre-plan which areas require high-resolution imaging and which can be imaged with reduced light intensity, thereby preventing unnecessary photobleaching in advance
Solution Approach 2:
The patent implements local quality by applying different light intensities to different regions of the sample. Regions identified as containing relevant image information in the overview image receive full STED light intensity for high-resolution imaging, while regions without relevant information receive reduced or no STED light intensity, minimizing photobleaching in those areas
2Measurement precision
If high light intensity is used to achieve increased spatial resolution, then spatial resolution is improved, but measurement time increases
Solution Approach 1:
The overview image generation serves as a preliminary action that quickly identifies regions of interest before the main imaging process. This allows the system to focus high-resolution STED imaging only on relevant areas, avoiding time-consuming full-sample high-resolution scanning and reducing total measurement time
Solution Approach 2:
The patent applies partial action by performing high-resolution STED imaging only on selected regions of interest identified in the overview image, rather than scanning the entire sample area at high resolution. This partial imaging approach maintains spatial resolution for relevant areas while significantly reducing measurement time
3Measurement precision
If full STED light distribution is used for scanning all sample segments, then spatial resolution is maintained, but unnecessary areas are exposed to high light intensities
Solution Approach 1:
The patent applies local quality by modulating the STED light intensity locally based on the overview image information. Sample segments containing relevant image information receive full STED light intensity to maintain spatial resolution, while segments without relevant information receive reduced or zero intensity, eliminating unnecessary energy exposure
Solution Approach 2:
The overview image generation performs a preliminary assessment of which sample segments contain relevant information. This preliminary action enables the system to pre-determine the light intensity distribution for the main imaging process, avoiding unnecessary energy expenditure on irrelevant areas
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 photobleaching and measurement time by minimizing exposure to high STED intensities, allowing for quicker and more efficient generation of high-resolution images while maintaining spatial resolution, and enabling the use of conventional mechanical scanners.
Implementation Method 1
a laser beam is focused into the sample to excite the fluorescent dyes present there to emit fluorescent radiation
Implementation Method 2
a focused de-excitation light distribution is superimposed on the excitation light distribution by irradiating light that de-excites the excited fluorescent dyes by stimulated emission
Data Source
Figure 1
Figure 2
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AI summary
The invention relates to a method for imaging a sample (18) using a fluorescence microscope (10) with stimulated emission depletion, comprising an imaging process, in which the fluorescence microscope (10) is controlled by means of a microscope control unit (42) in such a way that an illumination focus is generated, in which a focused excitation light distribution is superimposed with a focused de-excitation light distribution for stimulated emission depletion, as a result of which the sample (18) is excited during the illumination with the illumination focus only within an effective excitation focus, the extent of which is reduced in relation to the extent of the excitation light distribution, for the emission of imaging fluorescent light, and wherein, within a target region of the sample (18), a plurality of sample segments are scanned successively by means of the illumination focus with a first spatial resolution, which is matched to the reduced extent of the effective excitation focus, and are imaged in a corresponding plurality of image segments, from which a raster image (58) is generated. The fluorescence microscope (10) is further controlled by means of the microscope control unit (42) in such a way that prior to the imaging process an overview image (48) of the target region is generated with a second spatial resolution which is lower than the first spatial resolution and higher than a third spatial resolution which is adapted to the extent of the excitation light distribution, and the overview image (48) is analyzed for the identification of image regions without relevant image information. During the imaging process and the scanning of those sample segments, which are attributed to the image regions identified in the overview image (48) without relevant image formation, at least the radiation power of the de-excitation light distribution is reduced.