Structured Illumination Microscopy Reconstruction via Depth Information
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current microscopy methods face limitations in resolution due to diffraction limits, requiring multiple images at different phases and orientations, which increases measurement time and sample stress.
Innovation Solution
The method involves focusing a second focal plane and recording individual images at different illumination phases for both focal planes, using depth information from unfocused planes to reconstruct images without recording individual images from the additional plane, allowing for reduced focal plane spacing and shorter measurement times.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If multiple individual images are acquired at different phase positions and orientations to achieve superresolution, then the lateral resolution is improved, but the measurement time increases and the effective frame rate reduces
Solution Approach 1:
The patent applies partial action by acquiring individual images at only two phase positions (0 and π) instead of the conventional three or more phases, and by acquiring images at only two orientations (0° and 90°) instead of three or more orientations. This reduction in the number of measurements while maintaining superresolution capability directly addresses the contradiction by decreasing measurement time while preserving lateral resolution improvement.
2Measurement precision
If multiple individual images are acquired at different phase positions and orientations to achieve superresolution, then the lateral resolution is improved, but the stability requirement of the optical setup and sample increases
Solution Approach 1:
By reducing the number of phase positions and orientations at which images are acquired, the patent minimizes the duration of the measurement process. This shorter measurement window reduces the time during which optical drift or sample movement can occur, thereby lowering the stability requirements while still achieving superresolution through the partial sampling approach.
3Measurement precision
If the number of individual images is increased to achieve higher resolution, then the lateral resolution is improved, but the sample stress increases
Solution Approach 1:
The patent reduces sample stress by acquiring images at only two phase positions and two orientations instead of the conventional larger number of measurements. This partial sampling approach maintains superresolution capability while significantly reducing the total illumination dose and measurement time, thereby minimizing phototoxicity and sample stress.
4Productivity
If focal planes are spaced closer than half the minimum axial distance to reduce measurement time, then the productivity is improved, but the measurement precision according to Nyquist-Shannon sampling theorem is compromised
Solution Approach 1:
The patent creates virtual copies of focal plane information by using depth information from unfocused planes to reconstruct images of additional planes that were not directly imaged. This computational copying approach allows the system to achieve complete z-stack reconstruction with fewer physically spaced focal planes, thereby maintaining axial resolution while improving measurement speed and productivity.
Solution Approach 2:
The patent combines information from multiple sources (focused and unfocused planes) to create a composite reconstruction of the complete z-stack. By merging depth information from unfocused planes with structured illumination data from focused planes, the system achieves superresolution axial imaging with reduced focal plane spacing, overcoming the Nyquist-Shannon limitation while maintaining measurement 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
This approach reduces the number of focal planes needed, shortening measurement time and minimizing sample stress while maintaining high resolution, effectively violating the Nyquist-Shannon sampling theorem by incorporating 'apparent' focal planes in the reconstruction process.
Implementation Method 1
SIM is based on generating a spatial light structure on the sample under investigation, for example, by sinusoidal interference of the illumination light behind an optical grating
Implementation Method 2
The resolving power of microscopes depends on the aperture of the microscope objective and the wavelength of the light, due to the diffraction of light received from the sample in the microscope objective
Data Source
Figure 1
Figure 2~3
Figure 4~5
AI summary
In three-dimensional structured illumination microscopy (SIM), focal planes of the sample are focused, and each focal plane is sequentially illuminated in several phases with structured light. The sample light emitted by the sample is then captured as a separate image. From these images, a final image with increased resolution is reconstructed for each focal plane to generate a super-resolution image stack. This method is time-consuming and stresses the sample. By reconstructing a final image from individual images of two different focal planes using approximation techniques—an image representing a sample plane lying between these two focal planes—an image stack can be generated more quickly and with less stress on the sample.