Line-Scanning Microscope Structured Illumination Phase Control
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Solution Overview
Problem
Structured illumination microscopy faces challenges in achieving high resolution and contrast while minimizing out-of-focus signal noise, particularly when the structuring frequency approaches the diffraction-limited cut-off frequency, leading to reduced sample thickness and uneven enhancement in different spatial directions.
Innovation Solution
A line-scanning microscope design that uses two scanners to rotate and adjust the phase position of a structured periodic structure, combined with confocal detection, allowing for precise control of the scanning direction and phase position with minimal light loss, and enabling variable confocality.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If structured illumination is used to increase resolution and contrast, then resolution and contrast are improved, but out-of-focus signal increases leading to reduced signal-to-noise ratio
Solution Approach 1:
The detection process is segmented into multiple focal planes using z-stack acquisition. Each plane is detected separately with structured illumination, allowing out-of-focus signals from other planes to be excluded. The final image is reconstructed by combining only the in-focus signals from all segmented planes, thereby eliminating out-of-focus noise while preserving resolution enhancement.
Solution Approach 2:
Structured illumination employs periodic modulation of the illumination pattern (e.g., sinusoidal patterns at different phases and frequencies) to encode depth information. By analyzing the modulation transfer function across different spatial frequencies and phases, the system can distinguish in-focus from out-of-focus signals based on their different modulation responses, thereby improving signal-to-noise ratio while maintaining enhanced resolution.
2Object-affected harmful factors
If line structuring is used for confocal detection, then background signal is suppressed and contrast is maximized, but resolution enhancement is confined to one direction
Solution Approach 1:
The system merges wide-field structured illumination with confocal line-scanning detection. The line structuring provides confocal background suppression, while the structured illumination pattern (projected across the field) provides multi-directional resolution enhancement. By combining these approaches, the system achieves both background suppression and isotropic resolution improvement.
Solution Approach 2:
The system extends line structuring from one-dimensional scanning to two-dimensional structured patterns projected across the entire field of view. By using orthogonal line patterns or sinusoidal patterns in both x and y directions, the system achieves resolution enhancement in all spatial directions while maintaining confocal detection capabilities through temporal gating or z-stack acquisition.
3Ease of operation
If multiple separate actuators are used for controlling phase position and scanning, then precise control is achieved, but device complexity increases
Solution Approach 1:
A single galvanometer scanner is designed to perform multiple functions: it controls both the phase position of the structured illumination pattern and the scanning motion across the sample. By programming the scanner's deflection angles and timing, the system can generate different illumination patterns (line patterns, sinusoidal patterns) and perform both lateral scanning and axial sectioning, thereby reducing the number of required actuators while maintaining precise control.
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 enhances resolution and contrast uniformly across all spatial directions with low light loss, improving the signal-to-noise ratio and allowing for thicker sample imaging with reduced artifacts.
Implementation Method 1
the beam-shaping unit is composed of a combination of line-shaping optics (7) and a periodic structure (13)
Implementation Method 2
Different images with a different phase position of the periodic structure are generated by shifting the illumination structure
Implementation Method 3
scanner (9) is used to shift the line in the sample in the x-direction and scanner (23) to shift the line in the y-direction
Implementation Method 4
the light source (3), which is intensity-modulated by the modulator (5)
Data Source
Figure 1
Figure 2~3
Figure 4~5(b)
AI summary
The invention relates to a method and an assembly for the depth-resolved optical recording of a sample (29). According to said method: a sample or a portion thereof is scanned using linear illumination; the illumination of the sample at the focal point is periodically structured in at least one spatial direction; light originating from the sample is detected and images of the sample are generated; at least one sectional image and/or an image with an increased resolution through the sample is or are calculated from said images; images are acquired and the sectional image is calculated repeatedly by changing the orientation of the linear illumination of the sample and/or intervals are created on a flat panel detector (15) or a camera between lines in the illuminated sample area that is illuminated by the detection light, for line-by line non-descanned detection; and/or during a scanning operation a further light deviation across the line occurs in front of the detector in the scanning direction of the sample.