Depth-Resolved Luminescence Microscopy via Partially Overlapping Illumination Regions
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Solution Overview
Problem
Current luminescence microscopy methods are limited in achieving high-resolution imaging in the depth direction, as they either require complex interventions in the detection process or struggle to accurately determine the depth position of luminescent marking molecules.
Innovation Solution
The method involves introducing activation and/or excitation radiation into two regions that are perpendicular to the depth direction, with partial overlap, allowing for separate imaging of these regions to enhance depth resolution without altering the detection beam path, thereby improving the localization of luminescent marking molecules in the depth direction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional luminescence microscopy methods are used, then the imaging process is simple, but the depth resolution is limited and cannot accurately determine the depth position of luminescent marking molecules
Solution Approach 1:
The imaging process is segmented into multiple focal planes along the depth direction. The illumination system divides the sample into several discrete focal planes, and the detection system captures images from each plane separately. This segmentation allows precise depth localization of marking molecules by determining which focal plane they appear in, thereby improving depth resolution without requiring complex continuous depth scanning.
Solution Approach 2:
The method performs preliminary activation of marking molecules at specific focal planes before detection. By pre-activating molecules at known depth positions using selective plane illumination, the system establishes a depth reference framework beforehand. This preliminary action enables subsequent images to be accurately assigned to specific depth levels, improving depth measurement precision without adding complexity to the actual imaging process.
2Measurement precision
If multiple focal planes are used to improve depth resolution, then depth positioning accuracy increases, but the imaging time and number of images required increases
Solution Approach 1:
The illumination system employs periodic activation of marking molecules at different focal planes in a cyclic manner. Instead of continuously scanning through all depth planes, the system periodically illuminates and captures images from each focal plane in sequence. This periodic action reduces total imaging time by efficiently cycling through necessary depth planes while maintaining accurate depth positioning through the structured repetition of the imaging cycle.
3Measurement precision
If the detection beam path is altered to achieve high depth resolution, then depth imaging capability improves, but the system complexity and alignment requirements increase
Solution Approach 1:
The detection beam path is designed with multi-functionality to handle both standard wide-field imaging and depth-resolved imaging without requiring separate specialized pathways. The same detection optics and camera system are used for both functions, with the illumination system providing the depth discrimination capability. This universal design avoids the complexity of maintaining separate beam paths while still achieving high depth imaging capability.
Solution Approach 2:
The illumination system acts as an intermediary that provides depth information without requiring modifications to the detection beam path. By using selective plane illumination and activated marking molecules at different focal planes, the illumination side encodes depth information that is then captured by the standard detection system. This intermediary approach allows depth imaging capability to improve while keeping the detection beam path simple and unchanged.
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 imaging along the depth direction by reducing the spatial resolution to the size of the overlap region, allowing for precise determination of the depth position of luminescent marking molecules, thus overcoming the limitations of existing methods.
Implementation Method 1
activation and excitation of a subset of the marking molecules present in the sample to emit luminescent radiation
Implementation Method 2
marking molecules which can be activated in such a manner that, once activated, they can be excited to emit particular luminescent radiation
Data Source
AI summary
A method for high-resolution luminescence microscopy of a sample marked with marking molecules that can be activated to excite particular luminescent radiation, including: repeated activation of a subset of the marking molecules to emit luminescent radiation; repeated imaging of the sample along a depth direction and with a predetermined optical resolution; and producing images from the repeated imaging. Locations of the marking molecules are determined with a spatial resolution that is increased above the predetermined optical resolution. Activation of the marking molecules can be through radiation introduced into multiple regions, each extending along a plane substantially perpendicular to the depth direction. The regions can be arranged so that the regions are behind one another and overlap only partially. Separate images of the sample may be recorded for activation in each of the regions in order to obtain depth information relating to the marking molecules from the separate images.