Astigmatic Fluorescence Localization for Precise Sample Micromachining
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Solution Overview
Problem
Existing methods for localizing a region of interest in a sample using fluorescence microscopy are limited by resolution and accuracy in the on-axis direction, particularly when dealing with fluorescent entities fixed in orientation, leading to inaccuracies in micromachining processes like lamella production for electron microscopy.
Innovation Solution
Employing an astigmatic optical component in the fluorescence microscope to determine the position of fluorescent entities by evaluating astigmatism and ellipticity of the intensity profile, allowing for high-accuracy localization along the optical axis, and adjusting astigmatism based on focal plane depth and entity orientation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If a series of images focusing around the object of interest is acquired to obtain on-axis localization, then localization along the on-axis direction is achieved, but time is required and the fluorescent entity is prone to bleaching
Solution Approach 1:
The patent performs preliminary action by pre-calibrating the relationship between axial position and astigmatic intensity profile shape before actual localization. The astigmatic optical component is configured in advance to create predictable transformations, allowing single-shot 3D localization without requiring time-consuming series of focused images
Solution Approach 2:
The patent changes the optical parameter of the system by introducing astigmatism, which fundamentally alters how axial position information is encoded in the image. This parameter change enables extraction of z-position from a single 2D image intensity profile rather than requiring multiple images at different focal planes
2Measurement precision
If fluorescence microscopy is used to determine the position of fluorescent entities, then localization is achieved, but accuracy is limited for entities fixed in orientation
Solution Approach 1:
The patent uses asymmetry to overcome orientation limitations. The astigmatic transformation creates intensity profiles whose shape (ellipticity, orientation, and aspect ratio) depends on both axial position and molecular orientation. By analyzing the complete astigmatic pattern rather than assuming isotropic emission, the method can distinguish between position-induced and orientation-induced asymmetries, improving reliability for oriented entities
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
Achieves accurate localization of fluorescent entities with an accuracy of less than 50 nm, ensuring the region of interest is correctly positioned within the lamella for micromachining, thereby improving the precision of sample preparation for electron microscopy.
Implementation Method 1
the optics of the fluorescence microscope for imaging the sample onto a detector comprises an astigmatic optical component
Implementation Method 2
the region of interest comprises a fluorescent entity
Data Source
AI summary
A method and apparatus are provided for localization of a region of interest with a fluorescent entity inside a sample and for micromachining the sample in an integral fluorescence microscope/charged particle beam apparatus. The optics of the fluorescence microscope for imaging the sample onto a detector comprises an astigmatic optical component. The method comprises the steps of: determining a position of a focal plane of the fluorescence microscope with respect to a reference plane in the integral apparatus; obtaining an image of the fluorescent entity in the sample using the fluorescence microscope; determining a position of the fluorescent entity with respect to a focal plane of the fluorescence microscope, by evaluation of a degree of astigmatism and/or an ellipticity of a fluorescence intensity profile of the image of the fluorescent entity; and micromachining the sample around the determined position using a charged particle beam.