Conical Diffraction Microscopy for Sub-Diffraction Fluorophore Tracking
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Solution Overview
Problem
Existing microscopy techniques, including fluorescence microscopy, are limited by the diffraction limit, preventing the visualization of details smaller than 200-250 nm, which are crucial for understanding biological processes such as intracellular activities, protein folding, and DNA/RNA changes.
Innovation Solution
Conical diffraction microscopy (CODIM) uses optical singular distributions to overcome the diffraction limit by projecting light distributions with zero intensity at a common center, allowing for precise localization and tracking of biological objects beyond the Abbe's resolution limit through the Abbe's loophole technique.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional fluorescence microscopy is used, then the imaging process is simple and straightforward, but the resolution is limited by diffraction to 200-250 nm
Solution Approach 1:
The patent segments the imaging process into two distinct stages: a scanning phase that collects precise positional information through point-by-point measurement, and a reconstruction phase that builds the complete image from these localized data points. This segmentation allows achieving super-resolution without requiring complex optical hardware modifications.
Solution Approach 2:
The patent introduces temporal dimension by sequentially scanning different spatial locations and accumulating measurements over time. By adding this time dimension to the spatial measurement process, the system achieves resolution beyond the diffraction limit that would be impossible in a single static snapshot.
2Measurement precision
If the diffraction limit is accepted, then the optical system remains simple and widely applicable, but biological details smaller than 200-250 nm cannot be visualized
Solution Approach 1:
The patent performs preliminary localization measurements at each scanned position to precisely determine the centroid position of fluorescent emitters. This preliminary action of accurate point localization enables the subsequent reconstruction of super-resolved images with precision far exceeding the diffraction limit.
Solution Approach 2:
The patent creates multiple copies of the same biological structure by repeatedly scanning the same field of view multiple times. Each scan provides an independent measurement set, and by combining these copies through statistical reconstruction, the system achieves high precision localization that overcomes the diffraction barrier.
3Measurement precision
If repeated scanning is performed to improve localization precision, then measurement accuracy increases, but photobleaching and phototoxicity increase
Solution Approach 1:
The patent applies partial action by using just enough excitation light to achieve the required localization precision at each scan point, avoiding excessive illumination. The scanning approach concentrates light only on the regions of interest rather than illuminating the entire field continuously, thereby reducing overall photodamage while maintaining measurement accuracy.
Solution Approach 2:
The patent employs periodic scanning where the same field is scanned multiple times with intervals between scans. This periodic action allows fluorescent molecules to partially recover between excitation cycles, reducing cumulative photobleaching and phototoxicity while still accumulating sufficient data for high-precision localization through multiple measurements.
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
Enables high-resolution imaging and localization of biological objects with sub-diffraction limit precision, capturing details below 200 nm, enhancing understanding of biological processes and diagnostic capabilities.
Implementation Method 1
Conical diffraction microscopy (CODIM) uses optical singular distributions to overcome the diffraction limit by projecting light distributions with zero intensity at a common center
Implementation Method 2
allowing for precise localization and tracking of biological objects beyond the Abbe's resolution limit through the Abbe's loophole technique
Implementation Method 3
methods and apparatus for optical measurement, quantification and classification of biological objects using markers based on an inelastic interaction between the incident beam and the marker, such as, for example, fluorescent markers
Data Source
AI summary
A fluorophore recognition system performs a further function selected from the group consisting of tracking and imaging. The system includes: a light source that excites a fluorophore with a beam having a distribution with null intensity at a center thereof; a detector that measures photons emitted by the fluorophore; a processing unit that: measures fluorescence lifetime of the fluorophore; compares the lifetime with reference values associated with different fluorophores to identify the target fluorophore; and discriminates against spurious photons on the basis of lifetime. The processing unit also uses the emitted photons to guide performing the further function and minimize additional emissions. Recognition and the further function selected from tracking and imaging can be achieved using a set of fluorescence lifetime measurements in conjunction with discrimination against spurious photons.