Adjustable Beam Splitter for Microscope Spectral Unmixing
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Solution Overview
Problem
Current microscopy techniques face challenges in efficiently acquiring unique and meaningful spectroscopic data with minimal light loss, particularly when dealing with samples containing multiple fluorochromes with overlapping emission spectra.
Innovation Solution
The method involves using an adjustable beam splitter with a variable threshold wavelength in the scanning microscope's beam path to record signal intensity dependencies across multiple threshold wavelengths, allowing for efficient spectral division and data collection without light loss, and utilizing this information to determine the optimal setting for imaging and unmixing overlapping fluorochromes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If traditional fixed-wavelength beam splitters are used for spectral separation, then the microscope structure is simple, but light loss occurs and spectral flexibility is limited
Solution Approach 1:
The patent applies a dynamic beam splitter with adjustable threshold wavelength instead of a fixed-wavelength beam splitter. This allows the spectral separation point to be dynamically adjusted according to the specific fluorochromes being imaged, optimizing light distribution between channels while maintaining spectral flexibility without fixed structural constraints.
Solution Approach 2:
The invention changes the parameter of the beam splitter's threshold wavelength from fixed to adjustable. By varying this parameter across different imaging conditions and fluorochrome combinations, the system achieves optimal spectral separation efficiency while minimizing light loss, resolving the contradiction between adaptability and energy loss.
2Adaptability or versatility
If multiple fixed filters are used for spectral separation, then spectral coverage is limited, but device complexity increases
Solution Approach 1:
The adjustable beam splitter serves as a universal spectral separation element that can handle multiple fluorochrome combinations by simply adjusting its threshold wavelength parameter. This single multi-functional component replaces what would traditionally require multiple fixed filters and beam splitters, achieving broad spectral coverage without increasing device complexity.
Solution Approach 2:
Instead of using multiple fixed filters with different spectral characteristics, the invention uses a single beam splitter whose threshold wavelength parameter can be changed to match different spectral requirements. This parameter-based adaptability provides universal spectral coverage while keeping the device structure simple.
3Measurement precision
If signal intensity is recorded at multiple threshold wavelengths, then spectral data accuracy improves, but acquisition time increases
Solution Approach 1:
The system performs preliminary measurements at multiple threshold wavelengths to characterize the spectral signature of fluorochromes in the sample. This preliminary spectral data acquisition enables subsequent rapid imaging to be performed at optimized fixed threshold settings, achieving high measurement precision while minimizing actual imaging time through pre-characterization.
Solution Approach 2:
The method uses feedback from the signal intensity measurements at different threshold wavelengths to determine optimal imaging settings. By analyzing how signal intensity varies with threshold wavelength, the system identifies the best fixed threshold values for imaging, allowing rapid repeated measurements without requiring continuous time-consuming spectral scanning during actual imaging.
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 enables accurate, flexible, and cost-effective spectral data acquisition with high sensitivity, reducing light loss and improving image quality by allowing for precise spectral separation and unmixing of fluorochromes, while maintaining high efficiency and reproducibility.
Implementation Method 1
at least one adjustable beam splitter having an adjustable threshold wavelength, in particular a gradient filter, is arranged in the detection beam path
Implementation Method 2
light emitted from the sample is detected in at least one detection channel
Implementation Method 3
at least one adjustable beam splitter having an adjustable threshold wavelength, in particular a gradient filter
Data Source
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AI summary
The invention relates to a method for imaging a sample using a microscope, in particular a scanning microscope, in which the sample is illuminated with excitation light via an illuminating beam path, and light emitted from the sample is recorded via a detection beam path, wherein at least one adjustable beam splitter having an adjustable threshold wavelength is arranged in the detection beam path or/and in the illuminating beam path, and wherein light emitted from the sample is detected in at least one detection channel. According to the inventive method, for at least one predetermined sample region, a signal intensity of light detected in the at least one detection channel is recorded for a plurality of threshold wavelengths set at the adjustable beam splitter to obtain a signal/threshold-dependency of the predetermined sample region.