Two-Color Super-Resolution Microscopy Drift Correction
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Solution Overview
Problem
Existing two-color super-resolution fluorescence microscopy techniques face issues with channel crosstalk, background noise, and sample drift, which affect imaging quality and accuracy.
Innovation Solution
The method employs Alexa647 and Alexa750 or Cy5 and Cy7 fluorescent molecules with a customized imaging buffer solution and a real-time locking system to generate blinking fluorescent signals, separate emission spectra, and eliminate sample drift, thereby reducing channel crosstalk and background noise.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Speed
If fluorescent switches combining two fluorescent molecules with different excitation wavelengths are used for two-channel imaging, then quick switch between different imaging channels is realized, but severe channel crosstalk occurs and wrong co-localization information is produced
Solution Approach 1:
The patent separates the detection of two fluorescent molecules into distinct spectral bands using a spectral detector. Instead of using fluorescent switches that require sequential excitation at different wavelengths, the invention simultaneously detects emissions from both molecules across their respective spectral ranges, eliminating channel crosstalk while maintaining imaging speed.
Solution Approach 2:
The patent transitions from temporal separation (sequential channel switching) to spectral separation (simultaneous multi-wavelength detection). By using a spectral detector that captures the full emission spectrum, the system resolves overlapping signals in the spectral domain, eliminating crosstalk between channels.
2Adaptability or versatility
If ordinary fluorescent molecules with short excitation wavelengths are used, then two-channel imaging is achieved, but photo-bleaching occurs easily and autofluorescence background increases affecting imaging quality
Solution Approach 1:
The patent changes the excitation wavelength parameter to longer wavelengths (e.g., using Alexa 647 and Alexa 750) that reduce photo-bleaching and autofluorescence. Combined with spectral detection that can resolve emissions across the spectrum, this allows two-channel imaging using more stable fluorescent molecules with reduced background interference.
3Measurement precision
If fluorescent particles are added to samples for drift correction, then displacement can be recorded and subtracted, but difficult preparation is required and fluorescent particles occupy an imaging channel
Solution Approach 1:
The patent uses the biological sample's own fluorescent structures (e.g., nuclei, organelles) as drift reference markers instead of adding external fluorescent particles. The spectral detector captures emissions from both the drift references and the molecules of interest simultaneously, enabling drift correction without additional sample preparation or occupying imaging channels.
4Measurement precision
If fluorescent particles are used for drift correction, then displacement recording is possible, but fluorescence of particles attenuates over time due to photo-bleaching worsening correction precision
Solution Approach 1:
The patent employs endogenous fluorescent structures within the biological sample as drift references. These structures are naturally part of the sample and do not undergo photo-bleaching at the same rate as exogenous fluorescent particles. The spectral detector simultaneously monitors both the drift references and target molecules, maintaining correction precision throughout the imaging duration.
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 eliminates channel crosstalk, significantly reduces background noise, and ensures precise imaging by locking the sample in real-time, resulting in improved imaging quality and resolution.
Implementation Method 1
illuminating the biological sample by lasers to generate a blinking fluorescent signal of a first channel corresponding to the Alexa647 or Cy5 fluorescent molecules as well as a blinking fluorescent signal of a second channel corresponding to the Alexa750 or Cy7 fluorescent molecules
Data Source
AI summary
A two-color fluorescence localized super-resolution biological microscopy method and system are disclosed. The method includes: performing two-color fluorescence labeling on a biological sample by using Alexa647 and Alexa750 fluorescent molecules or Cy5 and Cy7 fluorescent molecules, and soaking the biological sample in an imaging buffer solution; illuminating the biological sample by laser to generate a flashing fluorescent signal of a first channel and a flashing fluorescent signal of a second channel respectively; constructing a super-resolution image of a first biological structure and a super-resolution image of a second biological structure respectively; and aligning the super-resolution image of the first biological structure and the super-resolution image of the second biological structure so as to construct a super-resolution image of a third biological structure. Thereby, the present method and system do not generate channel crosstalk and can significantly reduce background noise, so that the imaging quality is improved.