Three-Channel Fluorescence Microscope with Nanoscale Stabilization
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Solution Overview
Problem
Current super-resolution fluorescence localization microscopy faces challenges such as sample drift, difficulty in achieving multi-channel imaging, and inter-channel crosstalk, which affect imaging accuracy and efficiency.
Innovation Solution
A three-channel fluorescence localization super-resolution biological microscope system using a three-dimensional nanoscale stabilizing algorithm and simultaneous imaging with crosstalk removal algorithms, along with an optimized buffer solution for fluorescent dyes, to lock sample positions accurately and reduce imaging time.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If sequential imaging method is used to realize multi-channel super-resolution fluorescence microscopy, then different wavelengths of laser can be used to excite different fluorescent dyes, but the imaging time becomes long and imaging efficiency becomes low
Solution Approach 1:
The patent combines multiple imaging channels into a single simultaneous imaging process. By using a multi-channel fluorescent dye mixture and appropriate optical filtering, the system captures signals from different fluorescent dyes excited by different wavelength lasers at the same time, rather than sequentially imaging each channel separately.
Solution Approach 2:
The patent segments the detection process by using separate optical paths and detectors for each imaging channel. The multi-channel fluorescent dye mixture is excited by multiple wavelength lasers simultaneously, and the emitted signals are separated and detected independently through optical filtering and beam splitters, enabling parallel processing of multiple channels.
2Adaptability or versatility
If sequential imaging method is used, then multiple channels can be imaged one by one, but sample drift control becomes difficult and imaging accuracy decreases
Solution Approach 1:
The patent merges multiple imaging channels into a single simultaneous imaging process, capturing all channel signals at the same time point. This eliminates the time-dependent sample drift that occurs during sequential imaging, as all channels are recorded concurrently before any drift can occur.
3Measurement precision
If high intensity ultra-high imaging laser is used for one channel, then super-resolution imaging can be achieved, but erroneous excitation of other channels causes quenching of fluorescent dyes
Solution Approach 1:
The patent segments the excitation and detection processes by using multiple wavelength lasers with appropriate filters for each channel. Each laser wavelength is specifically filtered to excite only its target fluorescent dye, preventing cross-excitation and quenching while maintaining the high intensity needed for super-resolution imaging in each channel.
4Adaptability or versatility
If sequential imaging is used for multiple channels, then imaging can be performed one by one, but crosstalk among different channels affects imaging accuracy
Solution Approach 1:
The patent segments the optical detection paths for each channel using beam splitters and wavelength-specific filters. This physical separation ensures that signals from different fluorescent dyes and excitation wavelengths are routed to separate detectors, completely eliminating crosstalk between channels while maintaining simultaneous multi-channel imaging capability.
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 locking accuracy of 2 nm in the XY direction and 20 nm in the Z direction, allowing for deep cell imaging without crosstalk among channels, significantly reducing imaging time and improving resolution to 20 nm in the XY direction and 50 nm in the Z direction.
Implementation Method 1
a piezoelectric ceramic platform, and when the locking is begun, the locking program is arranged to control the piezoelectric ceramic platform through a piezoelectric ceramic platform drive to scan up and down on a longitudinal focal plane
Implementation Method 2
The laser illuminates the biological sample to respectively generate a first channel glittering fluorescent signal corresponding to the Alexa647 or Cy5 fluorescent molecules, a second channel glittering fluorescent signal corresponding to the Alexa750 or Cy7 fluorescent molecules and a third channel glittering fluorescent signal corresponding to the CF568 or Cy3B fluorescent molecules
Implementation Method 3
illumination light generated by the illumination module is reflected by a first multicolor reflector to illuminate a sample through the objective lens
Data Source
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AI summary
A three-channel fluorescence localization super-resolution biological microscope system includes a microscope main body, an illumination module (2), an imaging module (3) and a three-dimensional nanoscale sample locking module (4). The illumination module (2) is used to realize three channels, wide-field and semi-total internal reflection illumination and total internal reflection illumination and is adjustable in an illumination mode. Illumination light generated by the illumination module (2) illuminates a sample through an objective lens. Fluorescent light emitted from the sample is allocated by a light splitter (8) of a fixed ratio after passing through a first multicolor reflector (6) and a front imaging lens (7), one part is allocated into the three-dimensional nanoscale sample locking module (4), and the remaining part is allocated into the imaging module. A three-channel fluorescence localization super-resolution biological microscope method is disclosed.