Tiling Light Sheet Microscope for Simultaneous Dual-Color Imaging
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Solution Overview
Problem
Current light sheet microscopes face challenges in performing dual-color 3D fluorescence imaging efficiently due to fluorescence crosstalk and the need for sequential image acquisition, which doubles the imaging time, especially for high-resolution imaging of large biological tissues.
Innovation Solution
A tiling light sheet microscope with independent spatial light modulator assemblies for each laser wavelength range, allowing simultaneous dual-color imaging by generating and tiling excitation light sheets of different wavelengths independently, while maintaining spatial resolution and reducing image acquisition time.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If sequential dual-color imaging is performed using a dual-wavelength channel filter, then fluorescence crosstalk is reduced, but image acquisition time is doubled
Solution Approach 1:
The patent divides the imaging process into multiple light sheets (tiling) that are scanned across the sample. Each light sheet captures a portion of the image, and by scanning multiple light sheets sequentially, the system achieves both spectral separation and complete image coverage without requiring sequential acquisition of entire images at each wavelength
Solution Approach 2:
The patent implements continuous scanning of light sheets across the sample during illumination, maintaining continuous useful action by constantly moving the illumination and detection across the sample volume, thereby reducing total acquisition time while maintaining image quality
2Manufacturing precision
If tiling light sheet microscopy is used to achieve high spatial resolution, then imaging throughput is reduced due to the need to collect additional images
Solution Approach 1:
The patent introduces scanning in the lateral dimension by moving light sheets across the sample, transforming the imaging approach from purely axial stacking to a combination of axial and lateral scanning. This dimensional addition allows high-resolution imaging without requiring excessive numbers of axial slices, thereby improving throughput
3Measurement precision
If dual-color imaging is performed sequentially to avoid fluorescence crosstalk, then imaging time is doubled, but simultaneous imaging is difficult to achieve
Solution Approach 1:
The patent employs dynamic scanning of multiple light sheets across the sample during a single illumination cycle. The system dynamically adjusts the position and wavelength of light sheets in real-time, allowing simultaneous capture of multiple color channels by differentiating them through their temporal and spatial scanning patterns rather than requiring sequential acquisition
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
Enables simultaneous dual-color imaging without increasing image acquisition time, improving throughput by half compared to sequential methods, while maintaining high spatial resolution and reducing fluorescence crosstalk.
Implementation Method 1
a first spatial light modulator (SLM) assembly configured to perform modulation on the first laser beam. The tiling light sheet microscope further comprises a second spatial light modulator (SLM) assembly configured to perform modulation on the second laser beam
Implementation Method 2
a galvanometer mirror configured to direct the combined laser beams to an illumination path by offsetting an angle of the galvanometer mirror, and to generate, by scanning the laser beams, two types of excitation light sheets
Implementation Method 3
an excitation objective disposed at an end of the illumination path for illuminating a sample to be detected
Implementation Method 4
a detection objective configured to collect fluorescence emitted by the sample to be detected
Implementation Method 5
a combining optics configured to combine the first laser beam modulated with the first SLM assembly and the second laser beam modulated with the second SLM assembly
Data Source
AI summary
A tiling light sheet microscope and imaging method of a sample. The imaging method includes: generating a first laser beam of a first wavelength range and generating a second laser beam of a second wavelength range; performing a first optical modulation on the first laser beam and performing a second optical modulation on the second laser beam; and then combining the first laser beam and the second laser beam. The combined laser beams are directed onto an illumination path, to generate, by scanning the laser beams, a first excitation light sheet and a second excitation light sheet corresponding to the first wavelength range and the second wavelength range, respectively. The two types of excitation light sheets are combined in a propagation direction of excitation light so as to illuminate the sample. The fluorescences emitted by the sample are collected. In this way, for various biological tissues, at least dual-color simultaneous imaging can be performed, to keep same spatial resolution without increasing image acquisition time.