Dual Focal Plane Stabilization for Super-Resolution Microscopy
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Solution Overview
Problem
Super-resolution microscopy techniques face challenges in maintaining high accuracy due to sample drift, which can compromise image precision and accuracy, especially in three-dimensional imaging, where mechanical vibrations, temperature changes, and other mechanical movements cause significant issues, and existing drift correction methods are either offline or have limitations in real-time accuracy.
Innovation Solution
The implementation of a system that uses separate focal planes for imaging the sample and fiducial markers, allowing for real-time nanometer-scale drift correction by tracking the position changes of fiducial markers, such as microbeads or quantum dots, using the same objective lens, and employing actuators to correct for drift, ensuring that the sample is imaged without significant movement over extended acquisition times.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If super-resolution microscopy is used to achieve high resolution imaging, then lateral resolution can be better than 50 nm, but sample drift of 100 nm or more during acquisition destroys the high resolution nature of the image
Solution Approach 1:
The system continuously monitors the position of fiducial markers attached to the sample using a separate imaging system, calculates drift in real-time, and feeds this information back to actuators that adjust the sample stage position to compensate for drift, thereby maintaining image accuracy throughout the acquisition period
Solution Approach 2:
Fiducial markers serve as intermediary reference objects that are attached to the sample and tracked separately. These markers mediate between the sample position and the imaging system, allowing drift measurement and correction without interfering with the primary super-resolution imaging process
2Measurement precision
If SML methods are used to obtain super-resolution images, then exceptional high resolution can be achieved, but data acquisition times are extended to minutes or hours, placing even higher demands on minimizing sample drift
Solution Approach 1:
The system performs preliminary tracking of fiducial markers throughout the entire acquisition period and continuously corrects for drift in real-time, rather than attempting post-acquisition correction. This preliminary action ensures that even though acquisition times are extended to minutes or hours, the accumulated drift does not compromise the super-resolution quality
Solution Approach 2:
Real-time feedback from fiducial marker tracking allows the system to maintain correction accuracy throughout extended acquisition periods, enabling SML methods to achieve their full super-resolution potential without the time-induced drift that would otherwise degrade image quality
3Reliability
If conventional drift correction methods are used, then some correction can be achieved, but real-time nanometer-scale accuracy is not sufficient for deep imaging in super-resolution microscopy
Solution Approach 1:
The system separates the imaging function into two independent segments: a primary imaging system for super-resolution visualization and a separate tracking system for fiducial marker monitoring. This segmentation allows each system to be optimized for its specific function, with the tracking system achieving nanometer-scale precision independent of the primary imaging depth
Solution Approach 2:
The system adds a temporal dimension to drift correction by continuously monitoring fiducial marker positions throughout the acquisition period and applying real-time corrections, rather than relying on static or post-acquisition correction methods. This transforms drift correction from a static adjustment to a dynamic, time-resolved process
Data Source
AI summary
Methods and apparatus for deep microscopic super resolution imaging use two independent and variable focal planes. Movements of fiducial markers imaged using one focal plane are monitored and used to provide real-time or near real-time correction for sample drift. A second focal plane may be used to collect light for super-resolution imaging of a sample. A prototype embodiment has produced low drift when imaging many microns deeper than the fiducial markers.