Fluorescence Correlation Microscopy With Reference-Structure Beam Alignment
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Solution Overview
Problem
Fluorescence correlation spectroscopy (FCS) measurements are adversely affected by sample movement during prolonged measurements, leading to inaccurate results due to displacement of the excitation volume relative to the sample, especially in airy scan FCS configurations.
Innovation Solution
A microscopy method that uses existing sample structures as reference points to maintain the alignment of the excitation volume with the examination location, employing image evaluation methods and machine learning to correct positional deviations during the measurement process.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If FCS measurements are performed over extended time periods to improve measurement accuracy, then measurement precision is improved, but sample movement causes displacement of the excitation volume relative to the sample, worsening measurement reliability
Solution Approach 1:
The patent implements a feedback mechanism where the position of the excitation volume is continuously monitored relative to reference structures in the sample, and corrective actions are automatically applied to maintain proper alignment. This closed-loop control ensures that measurements remain reliable over extended time periods despite sample movement.
Solution Approach 2:
The patent introduces reference structures as intermediary elements that serve as stable positional markers. These reference structures act as mediators between the moving sample and the excitation volume, enabling continuous position monitoring and correction without directly interfering with the measurement process.
2Reliability
If the position of the excitation volume is continuously monitored and corrected to maintain alignment, then measurement reliability is improved, but device complexity increases due to additional monitoring and correction mechanisms
Solution Approach 1:
The patent designs the reference structures to serve multiple functions: they provide positional reference for alignment correction, serve as markers for measurement analysis, and can potentially indicate sample state changes. This multi-functionality reduces the need for separate dedicated components, thereby limiting the increase in device complexity.
3Manufacturing precision
If reference structures are introduced to maintain excitation volume positioning, then positioning accuracy is improved, but the measurement system becomes more complex requiring additional identification and tracking mechanisms
Solution Approach 1:
The patent enables the reference structures to automatically serve their positioning function without requiring complex external tracking systems. The reference structures inherently provide the necessary positional information, and the system leverages this self-service capability to maintain alignment with minimal additional complexity.
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
Ensures accurate and reliable FCS measurements over extended periods by continuously aligning the excitation volume with the desired examination location, reducing errors caused by sample movement.
Implementation Method 1
If molecules that are excitable to emit fluorescence radiation are present within the excitation volume, then these molecules are excited by the effect of the excitation radiation in the excitation volume to emit fluorescence radiation
Data Source
AI summary
A microscopy method involves directing a focused beam of excitation radiation at an examination location of an object to be examined, to create an excitation volume in the object. An overview image is used to identify at least one structure of the object and define at least one of the identified structures as a reference structure. A spatial relationship is defined between the positions of the examination location and the reference structure. Detection radiation coming from the excitation volume is acquired as measurement values over an overall measurement duration, the overall measurement duration being subdivided into a plurality of measurement intervals. At least every second measurement interval is preceded by a comparison of the current position of the focused beam with a current position of the examination location. The positioning of the focused beam is corrected in the case of an inadmissible deviation of the current positions.