Platinum Nanoprobe for Correlative Microscopy
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Solution Overview
Problem
Current imaging probes for biological samples in microscopy, such as those used in CLEM and immunocytochemical experiments, face challenges including difficulty in detection by TEM, requirement for costly and time-consuming silver or gold enhancement, toxicity, and instability under varying environmental conditions, which lead to artifacts and interference with biological samples.
Innovation Solution
The use of platinum nanoparticles with an oxidizing agent and an oxidizable substrate to generate an electron-dense osmiophilic precipitate, allowing for high-precision imaging without enhancement and maintaining stability across a wide range of conditions, while being functionalized with molecules for specific binding and fluorescence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Length of moving object
If quantum dots or quantum rods are used as imaging probes, then they can penetrate inside individual cells, but they are difficult to detect using TEM and require silver or gold enhancement which adds aspecific contribution
Solution Approach 1:
The patent combines fluorescent molecules with electron-dense nanoparticles (gold, silver, or platinum) to create hybrid probes that simultaneously provide fluorescence for optical microscopy and electron density for TEM detection, eliminating the need for separate enhancement steps
Solution Approach 2:
The imaging probes are designed to perform multiple functions: they can be detected by both fluorescence optical microscopy and transmission electron microscopy without requiring separate enhancement procedures, making them universally applicable for correlative microscopy
2Difficulty of detecting and measuring
If DAB osmiophilic polymers are produced by peroxidase-conjugated probes, then they can be detected by TEM, but the HRP enzyme cannot penetrate inside individual cells and is subject to denaturing
Solution Approach 1:
The patent uses small inorganic nanoparticles (gold, silver, or platinum) that can penetrate cells and replicate the electron-dense signal function of larger HRP enzymes, while being inherently stable under various environmental conditions including pH, temperature, and pressure variations
Solution Approach 2:
The patent changes the physical parameters of the probe by using smaller nanoparticle sizes (1-100 nm) compared to HRP enzymes, which enables cellular penetration while maintaining electron density for TEM detection and improving environmental stability
3Illumination intensity
If inorganic nanoparticles with luminescent ions are used, then they can be detected by fluorescence microscopy, but their large sizes can interfere with the biological sample
Solution Approach 1:
The patent reduces the size parameter of inorganic nanoparticles to 1-100 nm, which maintains sufficient fluorescence signal while minimizing interference with cellular structures and biological processes
Solution Approach 2:
The patent creates composite nanoparticles by coating small inorganic cores (gold, silver, or platinum) with fluorescent molecules or quantum dots, combining the advantages of both materials: small size for cellular penetration, electron density for TEM, and fluorescence for optical microscopy
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 significantly amplifies the signal in electron microscopy, enabling detection of nanoparticles as small as 1nm without enhancement, reduces toxicity, and allows for stable imaging across varying conditions, facilitating precise localization of molecules and reduced interference with biological samples.
Implementation Method 1
nanoparticles of platinum, an oxidizing agent and an oxidizable substrate capable of producing an electron-dense osmiophilic precipitate localized around the nanoparticles of platinum. The osmiophilic precipitate is obtained by oxidizing the oxidizable substrate activated by the nanoparticles of platinum and by the oxidizing agent
Implementation Method 2
The osmiophilic precipitate is obtained by oxidizing the oxidizable substrate activated by the nanoparticles of platinum and by the oxidizing agent
Implementation Method 3
The osmiophilic precipitate has a high electron-density that allows it to amplify the signal in electron microscopy and therefore make the nanoparticles of platinum clearly visible
Data Source
Figure 1~2
AI summary
Imaging method for a biological sample using microscopy, for example fluorescence optical microscopy, electron microscopy, or correlative microscopy, which provides to use imaging probes to obtain images in which it is possible to identify the imaging probes and/or possible molecules associated with them. The present invention also concerns the imaging probes, possibly functionalized, that can be used both in CLEM experiments and also in immunocytochemical experiments.