3D Localization Microscope Dual-Path Imaging
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Solution Overview
Problem
Current 3D localization microscopy methods face limitations in capture range, leading to long recording times and unwanted irradiation of fluorescence markers, as they can only detect and localize molecules within a small depth range of typically 1 to 1.5 μm, requiring multiple slices and resulting in inefficient imaging.
Innovation Solution
The method involves splitting the imaging beam path into two partial paths with different optical path lengths after the pupil, allowing for phase manipulation in the pupil plane to create a modified point spread function, enabling imaging across multiple focal planes and increasing the depth capture range by coordinating detectors with different focal planes, thus allowing for more accurate depth positioning of fluorescence emitters.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If the capture range is increased to reduce recording time, then productivity is improved, but measurement precision deteriorates due to the inability to accurately localize molecules outside the small depth range
Solution Approach 1:
The imaging system is segmented into multiple detection channels, each associated with a different focal plane. The beam path is split after the pupil into multiple partial beam paths with different optical path lengths, allowing simultaneous detection of fluorescence emitters at different depths. This segmentation enables the system to capture a larger depth range without sacrificing localization precision in any single plane.
Solution Approach 2:
The system transitions from single-plane 2D imaging to multi-plane 3D imaging by introducing depth as an additional detection dimension. By coordinating multiple detectors with different focal planes and using phase manipulation in the pupil plane, the system achieves accurate 3D localization of fluorescence emitters across an extended depth range, effectively adding a dimensional capability that resolves the contradiction between capture range and precision.
2Area of stationary object
If multiple slices are imaged to cover a larger depth region, then the measurement region is enlarged, but loss of time increases due to sequential imaging requirements
Solution Approach 1:
Multiple focal planes that would traditionally require sequential imaging are merged into a single simultaneous detection process. The beam splitting arrangement and phase manipulation enable all focal planes to be detected at the same time, combining the measurement capability for extended depth regions while eliminating the time loss associated with sequential slice acquisition.
Solution Approach 2:
The system maintains continuous useful action across the entire depth range by simultaneously detecting fluorescence emitters at all focal planes during a single exposure. This eliminates the interruptions and time losses inherent in sequential imaging, where the system would need to repeatedly focus and reimage different slices to cover the same depth region.
3Area of stationary object
If the illumination region is expanded to cover more of the specimen, then the measurement region is enlarged, but object-affected harmful factors increase due to unwanted irradiation of fluorescence markers
Solution Approach 1:
The system applies local quality by using plane-specific excitation and detection. Each focal plane has its own detection channel, allowing the system to selectively image and detect fluorescence emitters only in the intended focal region. This prevents unwanted irradiation and detection of out-of-focus markers, as the optical sectioning capability ensures that excitation and detection are localized to specific depth planes, reducing harmful photobleaching and background noise.
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 enhances the depth capture range, reducing unwanted irradiation and enabling the creation of high-resolution 3D images by accurately determining the depth position of fluorescence emitters across a broader region, facilitating more efficient and complete specimen imaging.
Implementation Method 1
phase manipulation in the pupil plane to create a modified point spread function
Implementation Method 2
fluorescence emitters in a specimen are repeatedly excited to emit fluorescent radiation
Data Source
AI summary
A microscope for high-resolution imaging of a sample in a depth direction and transversely thereto has an excitation beam path for illuminating a sample,—an imaging beam path with an objective and two detectors,—and a phase element. The phase element is situated in a pupil of the imaging beam path and has a different influence on two halves of the pupil cross section. The imaging beam path is split into two partial imaging beam paths downstream of the pupil as seen in the imaging direction, which partial imaging beam paths each lead to one of the two detectors. The two partial imaging beam paths have imaging lengths that differ by a specific wavelength difference such that the two detectors record images of the sample from two different focal planes, which are spaced apart by a distance in the depth direction.