Luminescence Microscopy Light Sheet Depth Resolution
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Solution Overview
Problem
In high-resolution luminescence microscopy, the excitation radiation bleaches marker molecules outside the image area, limiting the depth resolution and requiring numerous images to achieve a high-quality slice image, while determining the sample's position in the depth direction prolongs the measurement time and causes sample bleaching.
Innovation Solution
A method that excites a subset of marker molecules to emit luminescence radiation using a light sheet or overlapping light sheets, synchronizes the imaging with sample positioning, and uses a signal shaper to transmit triggering times and positions, allowing for precise control of the sample's vertical position during imaging to minimize bleaching and enable rapid depth-directional image recording.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If excitation radiation is applied to activate marker molecules in the entire sample volume, then luminescence signal is obtained from all depths, but marker molecules outside the image area are bleached and no longer available for further imaging
Solution Approach 1:
The sample volume is segmented into multiple depth layers using light sheet illumination at different positions. Each light sheet excites marker molecules only in its specific depth range, dividing the excitation process into spatial segments that prevent widespread bleaching while maintaining comprehensive coverage.
Solution Approach 2:
Excitation is applied locally rather than uniformly throughout the sample. Light sheets are positioned at specific depths and focused on particular regions, providing localized excitation that activates marker molecules only where needed, thereby preserving marker molecules in other regions for subsequent imaging.
2Loss of substance
If the sample is shifted in the depth direction during measurement to distribute bleaching, then sample bleaching is reduced, but determining the sample position for each image prolongs the measurement time
Solution Approach 1:
The measurement process uses periodic action by sequentially activating marker molecules in different depth layers through multiple light sheet positions. This periodic excitation pattern distributes the bleaching effect across different time periods and depth zones, reducing cumulative bleaching while maintaining efficient data acquisition.
Solution Approach 2:
Marker molecules are preliminarily activated in specific depth layers before imaging occurs. The light sheets pre-excite marker molecules in controlled sequences, ensuring that only the necessary subset of markers is activated at each stage, which reduces overall bleaching while preparing the sample for efficient imaging.
3Measurement precision
If many individual images are taken to achieve high-quality slice image with adequate depth resolution, then image quality is improved, but the total measurement time increases due to the large number of images required
Solution Approach 1:
The system transitions from two-dimensional lateral scanning to three-dimensional depth-resolved imaging by introducing light sheet positioning along the optical axis. This dimensional extension allows simultaneous capture of multiple depth layers, achieving high depth resolution without requiring sequential acquisition of numerous individual images.
Solution Approach 2:
The light sheet microscopy system performs multiple functions simultaneously: it provides optical sectioning, depth resolution, and wide-field imaging in a single measurement approach. This multi-functionality eliminates the need for repeated measurements at different depths, reducing total measurement time while maintaining high image quality.
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
Enables high-resolution, three-dimensional imaging without delaying the measurement process, reducing sample bleaching and allowing for any desired position profile in the depth direction, thus improving the efficiency and accuracy of luminescence microscopy.
Implementation Method 1
marker molecules, the marker molecules being excitable to emit luminescent radiation
Implementation Method 2
certain dyes, for example phosphors or fluorophores, are used in luminescence microscopy
Data Source
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AI summary
The invention relates to a method for high-resolution luminescence microscopy of a specimen marked with marker molecules, and to a luminescence microscope for performing the method, wherein the marker molecules can be excited to emit luminescence radiation. The method for luminescence microscopy comprises the excitation and imaging of marker molecules and the transmission of a trigger time and a position of the specimen. An optical recording device images the marker molecules in a capture area and transmits data from the imaging to an image capture circuit. The recording device transmits a time for the imaging to a signal former as a trigger time; the trigger time is then transmitted to a data recorder. The data recorder generates a position of the specimen at the trigger time and transmits said position to the image capture circuit, which links the position of the specimen in the depth direction to the data of the imaging of a frame such that a three-dimensional tomographic image of the specimen can be created.