Two-Photon Microscopy Signal Separation via Intensity Modulation
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Solution Overview
Problem
In two-photon excitation microscopes, high laser power is required to observe deep sample regions, leading to two-photon absorption at the surface, which results in out-of-focus fluorescence noise, and existing methods to exclude this noise decrease the efficiency of fluorescence generation at the focal point.
Innovation Solution
A method involving a pulsed illumination beam scanned over a sample, where signal light from non-linear optical processes is detected, and images are generated by acquiring mixed images including both in-focus and out-of-focus signals, then subtracting out-of-focus signals from these mixed images to isolate in-focus signals, without modulating the focus distribution, thus maintaining signal efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Length of stationary object
If laser beam power is increased to observe deep sample regions, then imaging depth is improved, but out-of-focus fluorescence noise increases due to two-photon absorption at the surface
Solution Approach 1:
The patent segments the total fluorescence signal into in-focus and out-of-focus components by acquiring multiple images with different laser beam powers. The in-focus signal is extracted through mathematical processing that separates the segmented signal components, allowing deep tissue imaging while removing surface-generated noise.
Solution Approach 2:
The patent changes the laser beam power parameter to acquire a series of images with different intensities. By varying this parameter and analyzing how the signal changes, the system can mathematically separate in-focus from out-of-focus fluorescence, enabling deep imaging with reduced noise.
2Object-generated harmful factors
If deformable mirror is used to modulate spatial distribution of laser beam at focal position, then out-of-focus fluorescence is reduced, but efficiency of fluorescence generation at focal position decreases
Solution Approach 1:
Instead of modifying the laser beam spatial distribution to suppress out-of-focus fluorescence, this patent extracts the out-of-focus component from the total signal through mathematical processing. By taking out the unwanted out-of-focus signal component computationally, the method avoids the energy loss that would result from optical modulation approaches.
Solution Approach 2:
The patent replaces the mechanical/optical approach (deformable mirror modulation) with a computational approach (mathematical separation of signal components). This substitution achieves noise reduction without the energy loss inherent in optical modulation methods.
3Measurement precision
If out-of-focus fluorescence signal is excluded by subtracting modulated image from original image, then signal purity is improved, but signal-to-noise ratio decreases
Solution Approach 1:
The patent performs preliminary acquisition of multiple images at different laser powers before attempting signal separation. This preliminary data collection provides the necessary information to subsequently extract both in-focus and out-of-focus components, preserving signal-to-noise ratio while achieving signal purity through mathematical processing.
Solution Approach 2:
The patent creates multiple copies of the sample image under different illumination conditions (different laser powers). By analyzing these copied images mathematically, the system can separate signal components and extract the in-focus fluorescence with preserved signal-to-noise characteristics.
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 allows for clear in-focus fluorescence imaging without decreasing the signal-to-noise ratio, as it effectively separates in-focus and out-of-focus signals without losing signal intensity from the focal point.
Implementation Method 1
signal light generated as a result of a non-linear optical process at each scanning position is detected
Implementation Method 2
a focal position of the illumination beam in the sample
Implementation Method 3
a pulsed illumination beam emitted from a light source is scanned while being focused at a sample
Data Source
AI summary
An image acquisition method in which a pulsed illumination beam emitted from a light source is scanned while being focused at a sample, signal light generated as a result of a non-linear optical process at each scanning position is detected, and an image of the sample is generated on a basis of the detected signal light, the image acquisition method including: acquiring a mixed image, which includes in-focus signal light generated at a focal position of the illumination beam in the sample and which also includes out-of-focus signal light; acquiring an image of the out-of-focus signal light on a basis of a plurality of mixed images having mutually different intensities of the out-of-focus signal light; and acquiring an image of the in-focus signal light by subtracting the image of the out-of-focus signal light acquired, from the mixed image acquired.