Charged Particle Beam Fluorescent Marker Localization
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing methods for localizing fluorescent markers, such as green fluorescent proteins (GFPs), in biological samples are limited by their ability to handle only small numbers of markers efficiently, requiring lengthy imaging times and suffering from statistical noise, which restricts their application to dense marker densities.
Innovation Solution
A charged particle beam system with optimized detector optics for simultaneous collection of secondary electrons and emitted light, combined with advanced image processing methods, enables the localization of large numbers of GFPs or quantum dots within minutes by selectively damaging markers and enhancing signal-to-noise ratios.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional light microscope techniques are used to image fluorescent markers, then the imaging process can be performed with standard equipment, but the imaging time becomes excessively long (tens of minutes) and statistical noise limits the ability to handle large numbers of markers
Solution Approach 1:
The patent combines a charged particle beam system with optical detection capabilities, merging the high-resolution positioning of the particle beam with the fluorescent marker detection capability. This hybrid system allows simultaneous acquisition of positional information and fluorescence signals, dramatically reducing imaging time from tens of minutes to minutes while handling large numbers of markers efficiently.
Solution Approach 2:
The patent replaces the conventional light microscope illumination and detection system with a charged particle beam system that can precisely locate fluorescent markers. The particle beam provides accurate spatial positioning while the system detects fluorescence emissions, substituting the slower optical scanning mechanism with a faster particle beam scanning approach.
2Measurement precision
If the charged particle beam scans the surface to damage markers and extinguish fluorescence for localization, then the position accuracy improves significantly, but the beam must be focused to a very small point which limits the number of markers that can be imaged simultaneously
Solution Approach 1:
The patent employs a scanning approach where the charged particle beam periodically visits different locations on the sample surface in a systematic pattern. This periodic scanning allows the system to methodically examine and localize numerous fluorescent markers across the entire field of view, overcoming the limitation of imaging only one marker at a time while maintaining high position accuracy.
3Measurement precision
If multiple laser flashes are used to excite fluorescent markers for localization, then the locations can be determined with sub-diffraction limit accuracy, but the process requires alternating with extinguishing laser flashes which extends the total imaging time to tens of minutes
Solution Approach 1:
The patent substitutes the laser-based excitation and detection system with a charged particle beam system that provides both positioning and detection functions. The particle beam replaces the need for alternating excitation and extinguishing laser flashes, achieving similar or better location accuracy while dramatically improving imaging throughput by eliminating the time-consuming laser alternation cycle.
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 precise localization of thousands of fluorescent markers in a minute, significantly improving the speed and accuracy of protein localization in biological samples, overcoming the limitations of prior art by enhancing signal collection and noise reduction.
Implementation Method 1
a charged particle apparatus and method capable of imaging samples containing fluorescent markers (FMs), such as green fluorescent proteins (GFPs) or quantum dots
Implementation Method 2
simultaneous collection of secondary electrons and emitted light
Data Source
AI summary
A method and system for the imaging and localization of fluorescent markers such as fluorescent proteins or quantum dots within biological samples is disclosed. The use of recombinant genetics technology to insert “reporter” genes into many species is well established. In particular, green fluorescent proteins (GFPs) and their genetically-modified variants ranging from blue to yellow, are easily spliced into many genomes at the sites of genes of interest (GoIs), where the GFPs are expressed with no apparent effect on the functioning of the proteins of interest (PoIs) coded for by the GoIs. One goal of biologists is more precise localization of PoIs within cells. The invention is a method and system for enabling more rapid and precise PoI localization using charged particle beam-induced damage to GFPs. Multiple embodiments of systems for implementing the method are presented, along with an image processing method relatively immune to high statistical noise levels.