Asymmetrical Light Sheet for 3D Localization Microscopy
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Solution Overview
Problem
Current 3D localization microscopy methods face limitations in depth resolution and efficient illumination, particularly with methods like PALM, which struggle to accurately determine the depth of fluorescent markers outside a predetermined range and often result in undesirable irradiation and limited activation cycles.
Innovation Solution
The method employs asymmetrical light sheet illumination, where the intensity distribution of the light sheet is offset from the focal plane, generating an axially dependent point spread function that allows for clear axial localization of fluorescent markers, enabling depth determination beyond the conventional depth range without unnecessary illumination.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If symmetrical light sheet illumination is used, then uniform excitation is achieved, but depth resolution and axial localization accuracy deteriorate
Solution Approach 1:
The patent applies asymmetry by deliberately offsetting the light sheet intensity distribution from the focal plane, creating an asymmetric illumination profile. This asymmetric illumination generates an axially dependent point spread function that enables clear depth resolution and axial localization of fluorescent markers, directly resolving the contradiction between uniform illumination and depth resolution.
2Reliability
If conventional PALM illumination is used, then activation of fluorescent markers is achieved, but unwanted irradiation occurs outside the focal range
Solution Approach 1:
The patent applies local quality by concentrating the light sheet intensity distribution at a specific offset position relative to the focal plane. This creates a localized excitation region with asymmetric intensity profile that activates fluorescent markers only within the desired axial range, preventing unwanted irradiation and photobleaching outside this region while maintaining activation efficiency.
3Illumination intensity
If light sheet is centered at the focal plane, then maximum brightness is achieved, but axial localization range is limited
Solution Approach 1:
The patent applies dimensionality change by shifting the light sheet intensity distribution from the focal plane position to an offset position along the axial dimension. This creates an asymmetric illumination profile that extends the effective axial localization range while maintaining sufficient brightness for detection, enabling observation of fluorescent markers beyond the conventional focal plane range.
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 depth resolution and reduces unwanted irradiation by analyzing the outline shape of isolated fluorescence markers, allowing for precise axial localization over a larger depth range without the need for filtering or overlapping light sheet areas.
Implementation Method 1
the excitation radiation is irradiated into the sample as a first light sheet which has an intensity distribution along the imaging direction that is asymmetrical to the focal plane
Implementation Method 2
the intensity distribution of the light sheet is offset from the focal plane, generating an axially dependent point spread function
Implementation Method 3
a sample is illuminated with excitation radiation to excite fluorescent markers in the sample to luminescence
Implementation Method 4
the sample is imaged into a single image by means of an imaging optic along an imaging direction, wherein the single image contains images of the luminescent fluorescent markers and the imaging optic has a focal plane and an optical resolution
Data Source
Figure 1~9
Figure 2~3
Figure 4~6
AI summary
A method for 3D high-resolution localization microscopy is described, in which: - in an excitation step, a sample (2) is illuminated with excitation radiation (4) to excite fluorescent markers in the sample (2) to luminescence; - in an imaging step, the sample (2) is imaged into a single image along an imaging direction (9) using imaging optics (3, 14), wherein the single image contains images of the luminescent fluorescent markers (11) and the imaging optics (3) has a focal plane (7) and an optical resolution; - the excitation and imaging steps are repeated several times to generate multiple single images, wherein the excitation steps are carried out such that at least for some of the luminescent fluorescent markers (11) their images are isolated in each of the single images; - in the generated,From several individual images of the isolated images of the luminescent fluorescent markers (11), a location of the corresponding fluorescent marker is determined with an accuracy exceeding the optical resolution; a high-resolution overall image is generated from the locations thus determined, whereby the excitation radiation is directed into the sample (2) as a first light sheet (4, 4.1) which has an intensity distribution (17, 17.1) along the imaging direction (5) that is asymmetrical with respect to the focal plane; and the isolated images of the luminescent fluorescent markers (11) in the individual images are analyzed with respect to their outline shape (13), and from the outline shape (13) a value about the distance of the corresponding fluorescent marker (11) from the focal plane (7) is derived.