Light Sheet Microscopy With Prism Reflectors for Isotropic Imaging
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Solution Overview
Problem
Existing light sheet microscopy systems face limitations in achieving sub-diffraction resolution and isotropic imaging over large volumes, particularly in thick samples like fruit flies and zebrafish embryos, due to issues like shadowing and striping artifacts, and require complex sample mounting procedures.
Innovation Solution
A light sheet microscopy system with multidirectional illumination capabilities using a single objective lens and multiple reflective surfaces, including prism reflectors, allows for isotropic structured illumination and improved axial resolution, enabling high-resolution imaging of thick samples with reduced hardware complexity and easier sample mounting.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Object-generated harmful factors
If multiple excitation objectives are used to vary light-sheet direction, then shadowing artifacts are reduced, but optical setup complexity increases
Solution Approach 1:
The patent divides the illumination function into multiple segments by using multiple reflectors (first reflector, second reflector, third reflector) positioned at different locations. Each reflector directs light from the single objective lens to illuminate different regions or angles of the sample, effectively segmenting the illumination coverage without requiring multiple objectives. This resolves the contradiction by maintaining simple optical setup (single objective) while reducing shadowing artifacts through multi-directional illumination.
Solution Approach 2:
The patent introduces reflectors as intermediary elements between the single objective lens and the sample. These reflectors act as mediators that redirect and redistribute light from the single objective to multiple illumination directions, eliminating the need for multiple objectives. The reflectors serve as the intermediary mechanism that enables multi-directional illumination with simplified optical architecture, thus reducing shadowing artifacts without increasing device complexity.
2Object-generated harmful factors
If light sheet is dithered quickly to provide partial relief, then shadowing is partially reduced, but imaging speed and resolution are compromised
Solution Approach 1:
The patent employs preliminary action by pre-positioning multiple reflectors at optimized locations before imaging begins. These reflectors are arranged to provide comprehensive multi-directional illumination coverage from the start, eliminating the need for dynamic dithering during acquisition. This preliminary configuration allows the system to achieve both shadowing reduction and high imaging speed simultaneously, as the illumination geometry is already optimized without requiring real-time adjustments that would limit frame rate.
3Device complexity
If single objective lens is used, then hardware complexity is reduced, but achieving isotropic resolution is difficult
Solution Approach 1:
The patent applies dimensionality change by introducing spatial reflection dimensions through multiple reflectors positioned at different locations and orientations. Instead of varying the objective lens itself (which would require multiple hardware components), the system creates multiple illumination directions by reflecting light through different spatial paths. This dimensional approach to light redirection enables isotropic resolution capability with a single objective lens, resolving the contradiction between hardware simplicity and imaging precision.
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 resolutions below 220 nm laterally and 0.2-10 μm axially, providing fast volumetric imaging at 0.5 to 5 volumes per second, suitable for neuroscience and developmental biology applications, while simplifying the imaging process and reducing costs.
Implementation Method 1
a beam steering mirror; a steering motor configured to move the beam steering mirror to each of two or more positions, wherein at each of the two or more positions the beam steering mirror is configured to steer the light sheet through the objective lens to a different selected reflector
Implementation Method 2
two or more reflectors each located in a different position in a sample plane that transects the sample volume orthogonal to the optical axis
Data Source
AI summary
Provided herein are microscopes providing multi-directional illumination of a sample volume by alternately reflecting a light sheet exiting a single objective lens off of multiple reflectors positioned about the sample volume. The provided microscopes exhibit high lateral and axial resolution and are particularly useful for imaging thick samples. Also provided are methods and computer instructions and systems for using the provided microscopes.