Fluorescent Marker Localization via Electron Beam Scanning
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Solution Overview
Problem
Conventional fluorescence microscopy has limited spatial resolution, typically restricted to around 500 nm, which is not sufficient for molecular-scale localization of markers in biological samples, and is influenced by environmental factors and marker lifetime.
Innovation Solution
A method involving a first radiation beam to excite markers and a second focused particle beam that scans across the sample to selectively damage and de-excite markers, allowing for improved spatial resolution by detecting the position where fluorescence flux decreases, using a focused particle beam with adjustable energy to cause damage and register its position.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional fluorescence microscopy is used to illuminate the sample with a light beam, then markers in the illuminated region are excited and fluorescence radiation is emitted, but the spatial resolution is limited to approximately 500 nm due to the size of the light beam focus
Solution Approach 1:
The patent segments the detection process into two distinct phases: first, excitation of markers throughout a larger volume using a light beam; second, selective detection from a smaller focal region using a focused electron beam. This segmentation allows the system to overcome the diffraction limit by separating the excitation volume from the detection volume, achieving molecular-scale spatial resolution while maintaining detection capability.
Solution Approach 2:
The patent introduces a focused electron beam as an intermediary detection mechanism. Instead of directly detecting fluorescence radiation from the light beam excitation, the electron beam serves as a mediator that scans through the sample, selectively exciting markers at its focal point and detecting their fluorescence. This intermediary approach enables super-resolution by confining the detection interaction to a volume much smaller than the initial excitation volume.
2Measurement precision
If the light beam focus is reduced to improve spatial resolution, then molecular-scale localization is achieved, but the wavelength constraints limit the minimum achievable focus size
Solution Approach 1:
The patent replaces the optical focusing mechanism (light beam) with an electron beam for the detection phase. Electron beams can be focused to much smaller dimensions than optical beams due to their shorter wavelength and stronger interaction with matter. This substitution allows achieving molecular-scale spatial resolution (nanometer scale) without being constrained by the wavelength of visible light, thereby decoupling spatial resolution from wavelength limitations.
3Reliability
If markers are excited continuously to maintain signal detection, then fluorescence radiation is continuously emitted, but marker lifetime is limited due to photochemical reactions
Solution Approach 1:
The patent employs periodic action by using a scanning electron beam that sequentially visits different locations in the sample rather than continuously illuminating the entire excitation region. The electron beam focuses on one location at a time, excites markers there, detects the fluorescence signal, then moves to the next location. This periodic scanning approach maintains continuous signal detection across the sample while significantly reducing the cumulative photochemical damage to individual markers, thereby extending their effective lifetime.
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 spatial resolution beyond conventional limits, enabling precise localization of markers to molecular scales and distinguishing between different types of markers, with the ability to perform 3D positional determination and simultaneous detection of multiple marker types.
Implementation Method 1
Irradiating the sample with a first radiation beam, as a result of which an excitation region is formed in the sample, in which excitation region markers are excited; Detecting fluorescence radiation emanating from markers in response to the excitation
Implementation Method 2
The energy of the particles in the particle beam is sufficient to lower the flux of fluorescence radiation emanating from markers impacted by the particles, as a result of damage
Data Source
AI summary
The invention describes a method of determining the position of fluorescent markers in a sample (4), with a high spatial resolution. To this end, the sample (4) is illuminated with an exciting light beam (11), while the sample (4) is simultaneously scanned by a particle beam (3). During scanning, markers will be impinged upon by the particle beam (3) and will be damaged, in such a manner that the marker impinged upon will no longer emit fluorescence radiation. This leads to a reduction of the flux of fluorescence radiation. This reduction is detected. Seeing as the position of the particle beam (3) w.r.t. the sample is known at the moment that the marker is damaged, the position of the marker in the sample is, accordingly, also known.