Metal Particle Markers for Optical Electron Microscope Alignment
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Solution Overview
Problem
Current methods fail to observe specimens in the same state using both optical and electron microscopes due to incompatibility of fluorescent markers with electron microscope preparation processes, leading to alignment issues between the two imaging modalities.
Innovation Solution
Utilizing metal particles that exhibit localized surface plasmon resonance, which act as markers in both optical and electron microscope images, allowing for precise alignment by estimating particle diameter from luminescent spot colors in optical images and confirming with electron microscope images.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fluorescent proteins or dyes are used for optical microscope observation, then localization of target cells or proteins can be observed, but the fluorescent markers lose fluorescence due to crosslinking by glutaraldehyde or oxidation by osmium tetroxide during electron microscope preparation
Solution Approach 1:
The patent introduces DAB (3,3'-diaminobenzidine) as an intermediary substance that converts the fluorescent signal into a permanent brown precipitate through enzymatic reaction. This mediator allows the optical microscope observation to be performed before electron microscope preparation, with the DAB reaction product serving as a stable marker that survives the harsh chemical treatments required for electron microscopy specimen preparation
Solution Approach 2:
The patent performs optical microscope observation with fluorescent markers before the specimen undergoes fixation and electron microscope preparation. The DAB reaction is performed in advance to create a permanent record of the fluorescent localization that can be correlated with subsequent electron microscope images, even though the original fluorescence is lost during preparation
2Reliability
If specimen dehydration and resin embedding are performed for electron microscope preparation, then the specimen can be observed with electron microscope, but shrinkage of the specimen occurs making alignment between optical and electron microscope images difficult
Solution Approach 1:
The patent uses DAB reaction products and metal particles as intermediary markers that are embedded in the specimen before dehydration. These markers serve as reference points that remain visible or detectable in both the optical microscope images (taken before dehydration) and electron microscope images (taken after dehydration), enabling alignment between the two imaging modalities despite the shrinkage that occurs during preparation
Solution Approach 2:
The patent creates a permanent copy of the fluorescent localization pattern through DAB reaction and metal particle deposition. This copied signal pattern serves as a reference that can be used to align the optical microscope image with the electron microscope image, even though the physical specimen has undergone shrinkage during preparation
3Reliability
If quantum dots are used as fluorescent dyes resistant to crosslinking and oxidation, then fluorescence signal can be maintained, but slicing the specimen into thin pieces for transmission electron microscope observation results in decrease in amount of fluorescent dye
Solution Approach 1:
The patent extracts the fluorescent markers (metal particles) into a separate, stable form that can withstand the specimen preparation process. By using metal particles that are deposited or embedded in the specimen, the system separates the optical detection function from the electron microscope compatibility requirement, allowing the markers to remain in the specimen throughout preparation without being lost during slicing
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 observation of specimens in the same state with both optical and electron microscopes, achieving precise alignment between the two imaging modalities by using metal particles as markers that are unaffected by electron microscope specimen preparation.
Implementation Method 1
preparing a specimen including, as a mark, a plurality of metal particles in which localized surface plasmon resonance is excited by irradiation with light
Data Source
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AI summary
An observation method includes: preparing a specimen including, as a mark a plurality of metal particles in which localized surface plasmon resonance is excited by irradiation with light; acquiring an optical microscope image by photographing the specimen with an optical microscope; acquiring an electron microscope image by photographing the specimen with an electron microscope; acquiring information of the positions and the colors of the plurality of metal particles in the optical microscope image; acquiring information of the positions and the particle diameters of the plurality of metal particles in the electron microscope image; and determining information for associating the optical microscope image and the electron microscope image based on the information of the positions and the colors of the plurality of metal particles acquired from the optical microscope image, and the information of the positions and the particle diameters of the plurality of metal particles acquired from the electron microscope image.