Bright-field Microscopy Phase Contrast via Defocus and DC Subtraction
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Solution Overview
Problem
In bright-field observation of colorless and transparent samples, such as cells, it is difficult to obtain clear images due to the lack of phase information, as the phase difference between zero-order and first-order diffracted light results in minimal contrast, making it challenging to observe samples accurately.
Innovation Solution
A sample observation method and device that involves acquiring an electronic image of a sample while the sample is displaced from the in-focus position, subtracting the DC component from the image, and adjusting the wavefront aberration to enhance contrast by aligning the first-order diffracted light with a specific wavefront aberration, allowing phase information to be obtained as contrast, thereby improving image clarity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If phase-contrast observation is used to observe colorless and transparent samples, then phase information can be obtained as contrast, but the device complexity increases due to the need for phase-contrast microscope and wavefront introduction means
Solution Approach 1:
The patent extracts only the necessary function of phase-contrast observation by removing the sample from the in-focus position, eliminating the need for complex phase-contrast microscope structures while retaining the ability to convert phase information into contrast
Solution Approach 2:
The patent changes the positional parameter of the sample relative to the in-focus position, using defocus to induce wavefront aberration that converts phase differences into intensity contrast, thereby achieving phase-contrast effect without phase-contrast microscope
2Ease of operation
If the sample is placed at the in-focus position for bright-field observation, then the observation is simple, but the contrast is insufficient making it difficult to observe colorless and transparent samples
Solution Approach 1:
The patent changes the positional parameter of the sample, placing it at a position displaced from the in-focus position to induce wavefront aberration, thereby converting phase information into intensity contrast and improving image contrast while maintaining bright-field observation simplicity
3Measurement precision
If the sample is displaced from the in-focus position to obtain phase contrast, then contrast information can be obtained, but the image quality deteriorates due to defocus
Solution Approach 1:
The patent uses image processing to detect and correct defocus blur, using the defocused image itself as feedback to restore image quality while preserving the contrast information obtained from the displaced sample position
Solution Approach 2:
The patent optimizes the displacement distance from the in-focus position to achieve the optimal balance between contrast enhancement and image quality, using wavefront aberration theory to determine the appropriate defocus amount
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 method enables clear observation of colorless and transparent samples by converting phase information into contrast, allowing for better visualization of the sample's details, even when the sample is not in the exact in-focus position, thereby improving image quality.
Implementation Method 1
When a sample is illuminated with parallel light flux, non-diffracted light (hereinafter, referred to as 'zero-order diffracted light') and diffracted light are generated from a sample
Implementation Method 2
A microscope, which makes an observation of the sample at a position displaced from the in-focus position of an image forming optical system so as to observe an image (phase-contrast image) in a wide observation field
Data Source
AI summary
A sample observation method includes an acquisition of for acquiring an electronic image of a sample, and a subtraction step of subtracting a DC component from a signal of the electronic image, and the acquisition step is performed in a state of bright-field observation, the electronic image at the subtraction step is an image acquired in a first predetermined state, and in the first predetermined state, at least a position of the sample and a in-focus position of an image forming optical system are different. A sample observation device includes a light source, an illumination optical system, an image forming optical system, an image-pickup device, and an image processing device, and the illumination optical system is disposed so as to irradiate a sample with illumination light from the light source, the image forming optical system is disposed so that light from the sample is incident thereon and an optical image of the sample is formed, the image-pickup device is disposed at a position of the optical image, and the image processing device is configured to implement the aforementioned sample observation method.