Graduated Filter Laser Scanning Microscope
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Solution Overview
Problem
Current Laser Scanning Microscopes face complexity in spectrally splitting light emitted from samples marked with multiple fluorescence dyes, requiring numerous fixed dichroic splitters or a cumbersome variable layout to optimally separate wavelengths, which limits their ability to efficiently detect individual dye emissions.
Innovation Solution
A flexible secondary beam splitter, such as a graduated filter, is introduced in the detection beam path, allowing continuous variation of its threshold wavelength, enabling adaptive spectral splitting without spatial separation of spectral parts, thus simplifying the arrangement and improving spectral resolution.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fixed dichroic splitters are used to separate wavelengths, then spectral separation is achieved, but device complexity increases due to numerous filters required
Solution Approach 1:
The patent applies parameter changes by using a tunable filter whose spectral characteristics can be dynamically adjusted. Instead of using multiple fixed dichroic splitters with different threshold wavelengths, the invention employs a single filter whose threshold wavelength can be changed to match different dye emission spectra, thereby reducing the number of physical components while maintaining spectral resolution
Solution Approach 2:
The patent implements universality by designing a detection system where a single tunable filter can serve multiple functions by adjusting its threshold wavelength to match different dye emissions. This multi-functional approach replaces the need for multiple specialized fixed filters, each designed for a specific wavelength range
2Measurement precision
If a variable layout is used to optimally separate wavelengths, then spectral resolution is improved, but ease of operation deteriorates due to cumbersome adjustment
Solution Approach 1:
The patent applies feedback by implementing a control system that automatically adjusts the tunable filter's threshold wavelength based on the detected dye emission spectrum. This closed-loop approach eliminates the need for manual layout adjustments while maintaining optimal spectral resolution, as the system self-regulates to match the appropriate filter characteristics
3Measurement precision
If multiple fixed dichroic splitters are used, then individual dye emissions can be detected, but adaptability decreases when examining different dyes
Solution Approach 1:
The patent applies parameter changes by using a tunable filter whose threshold wavelength can be dynamically adjusted to match the emission characteristics of different dyes. This allows the system to adapt to various dye types without requiring physical reconfiguration or replacement of filter components
Solution Approach 2:
The patent implements universality by designing a single detection system that can handle multiple dye types through the tunable filter's ability to adjust its spectral characteristics. This multi-functional design provides both precise detection capability and high adaptability across different fluorescence microscopy applications
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 solution allows for efficient and flexible spectral splitting of light, enabling better detection of individual dye emissions with reduced complexity and improved spectral resolution, facilitating the generation of high-quality three-dimensional images of specimens.
Implementation Method 1
The graduated filter NFT is located in the direction of detection behind the main dichroic beam splitter MDB, in particular in the beam path at the usual location for a secondary dichroic beam splitter SDS. The collimated initial incident beam is split by the graduated filter NFT in the direction of two detection beam paths DE1 and DE2.
Implementation Method 2
As a flexible secondary beam splitter NFT, a so-called graduated filter (for example short-pass) is used. This is a filter, the threshold wavelengths for the transmission of which varies in dependence of the position of the filter
Implementation Method 3
The light, which is emitted from the focal plane of the specimen and from the planes lying above and below it, reaches, passes through the scanner, to a dichroic beam splitter MDB. The latter separates the fluorescence light from the excitation light.
Implementation Method 4
After that, passing through a fiber or a suitable mirror arrangement, the laser beam reaches into the scanning module SM. After passing through the scanner, the laser beam generated in the light source is focused diffraction-limited on the specimen by the objective, the scanning optics and the tube lens.
Implementation Method 5
the laser beam generated in the light source is focused diffraction-limited on the specimen by the objective, the scanning optics and the tube lens
Implementation Method 6
In confocal detection (descanned detection) of fluorescence light, the light, which is emitted from the focal plane of the specimen and from the planes lying above and below it, reaches, passes through the scanner, to a dichroic beam splitter MDB. Thereafter, the fluorescence light is focused on a diaphragm (confocal diaphragm/pinhole) which is located exactly in a plane conjugate to the focal plane. As a result, parts of the fluorescence light beam outside the focus are suppressed.
Data Source
AI summary
Laser Scanning Microscope with an illumination beam path for illumination of a sample and a detection beam path for wavelength-dependent recording of the light from the sample, whereby filters for selection of the detection wavelengths are provided, characterized in that at least one graduated filter spatially variable in regard to the threshold wavelength between the transmission and reflection is provided in several partial beam paths for the selection of the wavelengths.