Microscope 3D Localization Using Microlens Array Sub-apertures
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Solution Overview
Problem
Current localization techniques in microscopy face challenges in precisely localizing fluorescent molecules in three spatial dimensions, especially in thick samples, due to shallow depth of field and overlapping images of molecules positioned closely along the z-axis, leading to low precision and long data collection times.
Innovation Solution
A microscope with illumination optics and a microlens array that subdivides the detection aperture, allowing for low-density point light source illumination and structured illumination to minimize image crossover, enabling precise 3D localization of individual molecules using light field technology and iterative algorithms for image reconstruction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If the density of fluorescent molecules is increased to improve structural precision, then more molecules can be localized, but the likelihood of image crossover increases, reducing localization precision
Solution Approach 1:
The detection aperture is segmented into multiple sub-apertures using a microlens array, allowing simultaneous capture of light field information from different spatial directions. This segmentation enables precise 3D localization of multiple molecules even when their images overlap in the final image plane, as each sub-aperture captures distinct angular information about the same molecular positions.
2Length of stationary object
If the depth of field is increased to detect molecules from a larger region, then more molecules can be localized in thick samples, but the precision of localization along the detection axis deteriorates
Solution Approach 1:
The patent transitions from 2D image plane coordinates to 3D light field coordinates by capturing images through multiple sub-apertures. Each sub-aperture provides angular information about the position of fluorescent molecules, enabling precise reconstruction of z-coordinates. The triangulation algorithm uses the spatial distribution of light across multiple sub-aperture images to determine the 3D position of each molecule, achieving high precision localization throughout the entire illuminated volume.
3Use of energy by moving object
If conventional optics are used to maintain high numerical aperture for light efficiency, then detection sensitivity is improved, but the depth of field remains shallow, limiting detection to a narrow region
Solution Approach 1:
The microlens array serves multiple functions simultaneously: it maintains the high numerical aperture of the detection objective for optimal light collection efficiency, while also creating multiple sub-aperture images that encode 3D spatial information. Each microlens acts as a separate aperture, and the combination of all microlens images provides both high sensitivity (due to high NA) and extended depth of field (through light field encoding).
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 allows for high-precision localization of molecules in three dimensions with reduced image overlap, enabling faster data collection and improved resolution in thick samples by minimizing the number of overlapping images and increasing the depth of field.
Implementation Method 1
a means for subdividing the detection aperture into individual sub-apertures is provided in a beam path of the detection optics such that images generated by the individual sub-apertures on the sensor of the camera depict an object volume from different spatial directions
Implementation Method 2
illumination optics for fluorescence excitation of point light sources of a sample
Data Source
AI summary
A microscope includes illumination optics for fluorescence excitation of point light sources of a sample, detection optics and a camera having a sensor. A density of the point light sources is kept low so as to minimize a crossover of point light sources that are behind or close to one another in each image captured by the camera. A means for subdividing a detection aperture into individual sub-apertures is provided in a beam path of the detection optics such that images generated by the individual sub-apertures on the sensor of the camera depict an object volume from different spatial directions.