Surface Plasmon Microscopy for Single Molecule Detection
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Solution Overview
Problem
Current methods for measuring single molecule activity within a living cell are difficult and time-consuming, hindering drug discovery and biological research, particularly in high content cellular screening where understanding drug effects within cells is crucial.
Innovation Solution
A surface plasmon assisted microscopy system is developed, utilizing a thin metal layer on a light translucent material with fluorophores capable of binding target analytes, where excitation light generates surface plasmons that enhance fluorescence emission detection at specific angles, allowing for precise detection of single molecules without degrading fluorophore emissions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional fluorescence microscopy is used to detect single molecules, then the detection can be performed with standard equipment, but the background noise is high and photobleaching occurs rapidly
Solution Approach 1:
A thin metal layer (50 nm or less) is introduced as an intermediary between the light translucent material and the fluorophores. This metal layer generates surface plasmons that mediate the interaction between light and fluorophores, enhancing fluorescence emission while reducing background noise and photobleaching through plasmonic field confinement
Solution Approach 2:
The system changes the physical parameters of light-matter interaction by utilizing surface plasmon resonance at specific angles. By controlling the excitation angle to match the surface plasmon angle, the system transforms conventional fluorescence excitation into surface plasmon-assisted excitation, achieving enhanced signal and reduced background
2Illumination intensity
If the metal layer thickness is increased to enhance surface plasmon effect, then the plasmon generation is improved, but the fluorophore emissions are degraded
Solution Approach 1:
The metal layer thickness is precisely controlled at 50 nm or less to optimize the balance between surface plasmon generation and fluorophore emission. This specific thickness parameter allows sufficient plasmon generation while maintaining transparency to fluorophore emissions, preventing excessive absorption or quenching
3Measurement precision
If high resolution single molecule detection is achieved, then spatial resolution is improved, but the optical sectioning becomes thick and background increases
Solution Approach 1:
The surface plasmon field is confined to a thin region near the metal layer surface, creating a localized excitation zone. This local field confinement ensures that only fluorophores within a thin optical section near the surface are excited, achieving high spatial resolution with reduced optical section thickness and minimal background from out-of-focus regions
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
The system achieves superior background rejection and reduced photobleaching, enabling the detection of single molecules in cells with enhanced spatial resolution and thin optical sectioning, making it more efficient and affordable for laboratories.
Implementation Method 1
an excitation source capable of exciting the one or more fluorophores, the excitation source positioned to strike the light translucent material at a first angle; and a light detector that detects emitted light generated by excited fluorophores at a second angle, wherein light emitted by the one or more fluorophores at the surface plasmon angle is detected through the microscope
Data Source
AI summary
The present invention includes a microscope and a method for using the microscope for single molecule with reduced photobleaching of a fluorophore (20) that includes a light translucent material (16); a metal layer (18) disposed on the light translucent material (16); a medium (15) disposed on the metal layer (18), the medium (15) having one or more fluorophores (20) capable of binding a target analyte (e.g., inside a cell); a microscope positioned to observe the surface plasmon emissions from the one or more fluorophores (20) within 50 nanometers of the surface of the metal layer (18); an excitation source capable of exciting the one or more fluorophores (20), the excitation source positioned to strike the light translucent material (16) at a first angle; and a light detector (38) that selectively detects emitted light generated by excited fluorophores (20) at a second angle (22), wherein light emitted by the one or more fluorophores (20) at the surface plasmon angle is detected through the microscope, such that single molecules may be detected without significantly degrading fluorophore (20) emissions.