Asymmetric Joint PSF Fiduciary Markers for 3D Drift Compensation
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Solution Overview
Problem
Current methods for compensating sample drift in fluorescence microscopy are limited by requiring additional optics, being costly, and failing to accurately track 3D positions, especially as imaging depth increases, leading to reduced precision and complexity in existing systems.
Innovation Solution
A method and system that utilize asymmetric joint point spread function distribution of fiduciary markers to estimate and correct for 3D sample drift without additional light sources, optics, or detectors, generating calibration curves for PSF width and lateral bias to calculate precise 3D positions and correct for drift during data acquisition.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If fiduciary marker based methods are used to track lateral positions, then drift compensation reliability is improved, but device complexity increases due to requirement of additional optics for axial tracking
Solution Approach 1:
The fiduciary markers themselves provide the information needed for 3D position tracking without requiring additional optical elements. The asymmetric joint point spread function distribution of the markers is analyzed to simultaneously determine both lateral and axial positions, making the system self-sufficient for complete drift compensation
Solution Approach 2:
The method changes from analyzing single PSF parameters to analyzing the joint point spread function distribution of multiple fiduciary markers. By examining the spatial distribution pattern of multiple markers and their individual PSFs, the system extracts both lateral and axial position information from the same optical signal without additional optics
2Measurement precision
If additional optical elements are added to track axial position of fiduciary markers, then measurement precision is improved, but device complexity and cost increase
Solution Approach 1:
The existing optical system's point spread function characteristics are utilized to provide axial position information. The asymmetric joint PSF distribution naturally encodes depth information that can be decoded through computational analysis, eliminating the need for specialized axial tracking optics
Solution Approach 2:
The method exploits the asymmetric nature of the joint point spread function distribution when multiple fiduciary markers are present at different axial positions. This asymmetry contains encoded information about the relative axial positions of markers, which can be extracted through computational analysis of the intensity distribution pattern
3Ease of operation
If image registration methods are used for drift compensation, then ease of operation is improved, but measurement precision deteriorates when number of labeled molecules is small
Solution Approach 1:
Fiduciary markers serve as stable intermediary reference points that are independent of the biological sample's labeled molecules. These artificial markers provide a reliable reference frame for drift compensation that does not depend on the density or stability of fluorescent labels in the sample
Solution Approach 2:
The method uses simple fluorescent or reflective fiduciary markers attached to the coverslip instead of complex hardware additions. These markers are inexpensive, easy to incorporate into the mounting protocol, and provide robust reference signals that persist throughout the imaging process
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 achieves high-precision single-molecule localization with motion blur limited to <2 nm laterally and <5 nm axially, applicable to both commercial and conventional microscopy systems, without increasing system complexity or cost, and maintains nanometer precision across the imaging process.
Implementation Method 1
the plurality of fiduciary markers have a joint point spread function distribution that is asymmetric
Implementation Method 2
Fluorescence microscopy is one of the most widely used techniques to probe nanoscale macromolecular interactions
Data Source
AI summary
A method of estimating relative change of the 3D position of an object (e.g., sample drift in a microscopy system) having fiduciary markers that have an asymmetric joint point spread function distribution includes generating a plurality of calibration curves for each of the markers during a calibration phase including first calibration curves for a PSF width and second calibration curves for lateral bias. The method further includes capturing a first image of the markers during a data acquisition phase, generating a first joint 3D position for the markers using the first image, the first calibration curves and the second calibration curves, capturing a second image of the markers during the data acquisition phase, generating a second joint 3D position for the markers using the second image and the first and second calibration curves, and estimating the sample drift using the first joint 3D position and the second joint 3D position.