Inclined Light Sheet Microscopy via Pupil Offset
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Solution Overview
Problem
Conventional light sheet microscopy systems face challenges such as mechanical complexity, high cost, limited detection efficiency, and reduced flexibility due to the need for mechanical movements and multiple optical setups, which hinder efficient imaging and sample orientation.
Innovation Solution
A method and device that utilize a light sheet inclined at an angle other than 90° with the optical axis, where the illumination light is offset into the entrance pupil of the objective, allowing for adjustment of the entry point on a pitch circle and changing the irradiation direction, enabling two- or three-dimensional imaging with light field technology and eliminating the need for mechanical movements and additional optical setups.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If the light sheet is inclined at an angle other than 90° to the optical axis and the area detector is moved synchronously with the rotation of the illumination plane, then the illuminated sample area can be continuously tracked, but the device complexity increases due to synchronous mechanical movement requirements
Solution Approach 1:
The patent replaces the mechanical synchronous movement system with an optical solution using a tube lens and prism rotation. Instead of physically moving the area detector in sync with the illumination plane rotation, the system rotates a prism in the detection optical path to redirect light onto a stationary detector, substituting mechanical complexity with optical redirection.
Solution Approach 2:
The patent introduces a tube lens and rotating prism as intermediary optical elements between the sample and the area detector. These intermediaries enable the detection of the illuminated sample area without requiring the detector itself to move, thus decoupling the detector position from the illumination plane orientation.
2Adaptability or versatility
If the angle between the illuminated plane and the detection direction is changed, then different sample orientations can be imaged, but the fulfillment of the Scheimpflug condition becomes impossible for standard imaging ratios
Solution Approach 1:
The patent changes the optical parameters by introducing a tube lens with a specific focal length and rotating a prism within the detection optical path. This allows the system to maintain proper focus and imaging geometry (Scheimpflug condition) across different illumination angles by adjusting the prism rotation angle and tube lens positioning, rather than being constrained by fixed standard imaging ratios.
3Productivity
If multiple detection objectives and tube lens systems are used to image oblique target areas, then the illuminated sample area can be captured, but the detection efficiency is limited due to the inclination of various optics
Solution Approach 1:
The patent merges the illumination and detection functions into a single objective lens, eliminating the need for separate detection objectives. The inclined light sheet illuminates the sample, and the same objective collects the fluorescence signal, which is then redirected by a rotating prism onto a stationary area detector, simplifying the optical path and improving detection efficiency.
Solution Approach 2:
The patent replaces the complex multi-objective detection system with a single objective combined with a rotating prism mechanism. This substitution reduces the number of optical interfaces and alignment requirements, thereby improving light transmission and detection efficiency while maintaining the ability to capture oblique sample areas.
4Measurement precision
If the objective has a depth of field smaller than the sample volume depth, then higher resolution imaging is achieved, but the entire sample volume cannot be imaged in a single focal plane
Solution Approach 1:
The patent employs periodic scanning of the inclined light sheet through the sample volume by rotating the illumination plane around the optical axis. This periodic action allows different focal planes at different depths to be sequentially illuminated and imaged, enabling comprehensive 3D coverage of the entire sample volume while maintaining high resolution in each focal plane.
Solution Approach 2:
The patent adds the temporal dimension to the imaging process by sequentially scanning through different depths of the sample volume. Instead of attempting to image all depths simultaneously (which would compromise resolution), the system captures multiple 2D focal planes at different depths over time, reconstructing the complete 3D sample volume from these sequential 2D images.
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 enhances imaging efficiency, reduces costs, and increases flexibility by allowing standard lenses to be used effectively, with improved depth of field and detection efficiency, and enables imaging of sample volumes beyond the focal plane without mechanical tracking of the detection surface.
Implementation Method 1
light sheet microscopy illuminates a thin layer in the sample, corresponding to the thickness of a light sheet, allowing the measurement and visualization of an optical section
Implementation Method 2
The fluorescent radiation originating from the target area illuminated by the light sheet is captured by the objective and directed into a detection optics system
Data Source
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AI summary
The invention relates to a method and an arrangement for light-sheet microscopy. The arrangement comprises: - an objective (1) and means for scanning (4) a sample volume (10) to be imaged with illumination light shaped into a light sheet (9) which forms an angle δ ≠ 90° with the optical axis (8) of an objective (1), wherein - the light sheet (9) is aligned in a propagation direction into the entire sample volume (10) to be imaged, and wherein - the objective (1) has a depth of field Sobj in the direction of the optical axis (8) which is less than the depth T of the sample volume (10), - the objective (1) serves both for illumination and detection, and - the illumination light is irradiated into the entrance pupil (24) of the objective (1) at an entrance point (23) offset parallel to its optical axis (8).and - an optical device downstream of the lens (1) for generating two-dimensional or three-dimensional images of the sampled sample volume according to light-field technology, - a spatially resolving optoelectronic area sensor (17) downstream of the optical device, and - hardware and software configured for generating images of the sample volume (10) from the electronic image signals emitted by the area sensor (17).