Fluorescence Microscope Dual Illumination Switching SIM and LM
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Solution Overview
Problem
Fluorescent microscopes face challenges in achieving high-resolution imaging due to the diffraction limit, particularly in combining structured illumination microscopy and localization microscopy, as they often require either low intensity illumination or a small illuminated region, limiting the size of the sample area and acquisition time.
Innovation Solution
A fluorescence microscope with a dual illumination system that switches between structured illumination and localization illumination modes, using a pattern generation system to spatially modulate light and an image detector to capture high-resolution images, allowing for simultaneous imaging of large sample areas with improved intensity and precision.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If structured illumination light is used to illuminate the sample, then the resolution is improved beyond the diffraction limit, but the intensity of the illumination light is substantially reduced
Solution Approach 1:
The patent combines structured illumination microscopy (SIM) and localization microscopy (LM) into a single optical setup with a dual illumination system. The switching mechanism alternates between SIM illumination (for resolution improvement) and LM illumination (for high intensity), merging the advantages of both techniques to achieve high resolution while maintaining sufficient illumination intensity for photon collection.
2Measurement precision
If the size of the illuminated region is reduced to achieve high intensity illumination, then the localization precision is improved, but the area of the sample that can be imaged is limited
Solution Approach 1:
The patent implements dynamic switching between two illumination modes: SIM mode with a larger illuminated region for contextual information, and LM mode with a reduced illuminated region for high precision localization. The switching mechanism dynamically alternates between these modes, allowing the system to adapt the illuminated region size based on the current imaging requirement, thus achieving both large area coverage and high localization precision.
3Adaptability or versatility
If a switching mechanism is added to alternate between SIM and LM illumination modes, then the versatility of the microscope is improved, but the device complexity increases
Solution Approach 1:
The patent designs a universal illumination system that can perform both SIM and LM functions using shared optical components. The switching mechanism directs illumination light through different optical paths (SIM optical path or LM optical path) that converge on the same sample stage and detection system. This multi-functional design allows a single microscope to execute both imaging techniques without requiring completely separate systems, thereby improving versatility while controlling complexity.
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 high-resolution three-dimensional imaging with improved precision and accuracy by combining structured illumination microscopy and localization microscopy in a single optical setup, enhancing the resolution and localization precision by a factor of 3 to 4 compared to conventional methods.
Implementation Method 1
If the system for generating structured illumination light is an interferometric system
Implementation Method 2
This light interacts with fluorophores in the sample which then emit light of a different wavelength
Implementation Method 3
Due to the diffraction, point objects are seen as blurred disks (called Airy disks) surrounded by diffraction rings
Data Source
Figure 1a~1c
Figure 2a~3c
Figure 4
AI summary
The invention relates to a fluorescent microscope and a respective method for obtaining super-resolution images of a sample labelled with at least one type fluorescent label by combining localization microscopy and structured illumination microscopy. In an aspect, the fluorescent microscope comprises one or more light sources and an illumination system having a structured illumination path, in which a pattern generation system is positioned, for illuminating the sample with structured illumination light and a localization illumination path for illuminating the sample with localization illumination light. A switching mechanism is configured to switch between a first, a second and/or a third mode, wherein in the first mode at least a portion of the light emitted from the one or more light sources propagates through one of the illumination paths; in the second mode at least a portion of the light emitted from the one or more light sources propagates through the other one of the illumination paths; and in the third mode at least a portion of the light emitted from one or more of the light sources propagates through one illumination path while simultaneously at least another portion of the light emitted from one or more of the light sources propagates through the other illumination path. At least one image detector positioned in an optical detection path, configured to detect at least a portion of fluorescent light emitted from fluorescent molecules of the illuminated sample. Another aspect concerns a method for obtaining super-resolution image data of a sample labeled with at least one type of fluorescent label comprising illuminating the sample with localization illumination light and with structured illumination light; detecting at least a portion of the fluorescent light emitted from the excited fluorescent molecules of the at least one fluorescent label, thereby obtaining at least one image of the illuminated sample; and processing the obtained at least one image of the sample image to obtain super-resolution image data.