Structured TIRF Microscopy Interference Pattern Blur Rejection
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Solution Overview
Problem
Existing TIRF and HILO microscopy techniques face challenges in rejecting out-of-focus blur, leading to reduced image contrast and reconstruction artifacts, particularly in densely labeled cells and when evanescent fields are compromised by light scattering.
Innovation Solution
The implementation of a structured illumination TIRF (sTIRF) and HILO microscopy system using two mutually coherent beams of light, where the interfering beam creates interference with the primary beam, allowing for the detection of static or fluctuating fluorescence emissions to differentiate in-focus from out-of-focus regions, thereby generating a final image with improved contrast.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If evanescent fields are used in TIRF microscopy to achieve high signal to background ratios, then sensitivity is improved, but light scattering causes photons to propagate into the far field resulting in out-of-focus blur
Solution Approach 1:
The patent segments the illumination into two distinct coherent beams that create an interference pattern, allowing spatially selective excitation. This segmentation enables the system to distinguish between in-focus and out-of-focus regions by analyzing fluorescence fluctuations across multiple images with different interference pattern positions, thereby rejecting out-of-focus blur while maintaining high signal-to-background ratios.
Solution Approach 2:
The patent employs periodic modulation of the interference pattern by laterally shifting it across the field of view during image acquisition. By capturing multiple images at different interference pattern positions and analyzing the periodic fluorescence fluctuations, the system can identify and reject out-of-focus signals that do not exhibit the expected periodic modulation, thus eliminating blur while preserving sensitive detection.
2Volume of moving object
If HILO microscopy uses shallow angle illumination to interrogate the entire cell volume, then imaging depth is improved, but out-of-focus excitation is inevitable degrading image quality
Solution Approach 1:
The patent segments the illumination field using coherent interference to create a structured pattern that can be laterally positioned. This allows the system to selectively illuminate specific regions within the cell volume at a time, reducing out-of-focus excitation compared to uniform shallow angle illumination, while still enabling three-dimensional sampling by moving the interference pattern across different depths.
Solution Approach 2:
The patent replaces mechanical z-axis scanning with optical sectioning using coherent interference patterns. Instead of physically moving the focal plane through the cell volume, the system uses the interference pattern's lateral displacement to selectively excite different depths, achieving volumetric imaging with reduced out-of-focus excitation through optical rather than mechanical means.
3Manufacturing precision
If two-dimensional structured illumination microscopy is combined with TIRF, then resolution is improved, but reconstruction artifacts occur when out-of-focus blur is present
Solution Approach 1:
The patent uses periodic lateral shifting of the interference pattern to modulate in-focus fluorescence signals, creating characteristic fluctuations that can be distinguished from out-of-focus signals during reconstruction. This periodic modulation provides a reliable signature for identifying valid in-focus data, enabling artifact-free reconstruction even in the presence of out-of-focus blur.
Solution Approach 2:
The patent implements a feedback mechanism where fluorescence fluctuations detected during interference pattern scanning are used to identify and reject out-of-focus regions. By analyzing the temporal modulation of fluorescence signals across multiple images, the system provides feedback to the reconstruction algorithm to exclude contaminated data, ensuring high reconstruction accuracy without artifacts.
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 effectively rejects out-of-focus blur, resulting in higher contrast images with enhanced optical sectioning capabilities, improving image quality and detail in both TIRF and HILO microscopy.
Implementation Method 1
The interfering beam of light may be configured to create interference with respect to the primary beam of light
Implementation Method 2
Total Internal reflection fluorescence (TIRF) microscopy has become an indispensable tool in cell biology. By combining widefield detection with high numerical aperture lenses and optical sectioning with evanescent fields
Implementation Method 3
detection of whether fluorescence emissions from the sample being imaged fluctuate or remain static
Data Source
AI summary
Systems, methods, and computer-readable storage media are disclosed for providing a structured total internal reflection fluorescence (sTIRF) imaging system providing improved out of focus blur rejection and improved image contrast. The sTIRF imaging system may be configured to illuminate a sample using two beams of light (e.g., a primary beam of light and an interfering beam of light). The interfering beam of light may be configured to create interference with respect to the primary beam of light. The sTIRF imaging system may be configured to capture a plurality of intermediate images of the sample during the illuminating, and to generate a final image of the sample based on the plurality of intermediate images. The interference caused by the two beams of light may enable the sTIRF imaging system to reject out of focus blur based on detection of whether fluorescence emissions from the sample being imaged fluctuate or remain static.