Microscope Fluorescence Noise Reduction via Dynamic Pinhole Positioning
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Solution Overview
Problem
Conventional microscopes face difficulties in distinguishing in-focus fluorescence from out-of-focus fluorescence, especially when observing samples at depth, due to out-of-focus fluorescence leaking into the confocal pinhole, which affects image clarity and precision.
Innovation Solution
A microscope system that uses a light-blocking member, such as a pinhole, to selectively transmit in-focus fluorescence while blocking out-of-focus fluorescence by adjusting the positional relationship between the light-blocking member and the focal point, allowing for the detection of both types of fluorescence at different times and calculating their difference to remove noise.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If a confocal pinhole is used to block out-of-focus fluorescence, then image clarity is improved, but the ability to observe samples at depth is reduced due to out-of-focus fluorescence leaking into the pinhole
Solution Approach 1:
The patent segments the fluorescence detection process into two distinct temporal phases: a first detection phase where the light-blocking member is positioned to allow in-focus fluorescence to pass through, and a second detection phase where the light-blocking member is positioned to block in-focus fluorescence. This temporal segmentation allows separate measurement of in-focus and out-of-focus fluorescence components, which are then combined to produce a corrected image that maintains clarity while accurately representing deep sample structures.
2Illumination intensity
If out-of-focus fluorescence is allowed to pass through the pinhole, then signal intensity is improved, but image precision deteriorates due to noise from out-of-focus fluorescence
Solution Approach 1:
The patent implements a feedback mechanism where out-of-focus fluorescence is first detected separately (when the light-blocking member blocks in-focus fluorescence), and this detected out-of-focus signal is then used to correct the in-focus fluorescence image. The arithmetic operation unit subtracts the out-of-focus fluorescence component from the total fluorescence signal, providing feedback correction that removes noise while preserving the useful in-focus signal intensity.
3Device complexity
If the light-blocking member position is fixed, then device complexity is reduced, but the ability to distinguish in-focus from out-of-focus fluorescence is limited
Solution Approach 1:
The patent introduces dynamic positioning of the light-blocking member along the optical axis, allowing it to switch between at least two different positions. This dynamic adjustment enables the system to alternately achieve optically conjugate and optically nonconjugate relationships between the light-blocking member and the focal point, thereby enabling temporal separation of in-focus and out-of-focus fluorescence detection without requiring complex spatial arrangements.
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 the signal-to-noise ratio, resulting in sharper images with reduced noise, particularly effective for high-precision observations and samples with scattered substances, and allows for accurate fluorescence imaging even with moving samples.
Implementation Method 1
an objective optical system that focuses the excitation light scanned by the scanner onto a sample and that collects fluorescence generated by the sample at each scanning position
Implementation Method 2
a setting unit that sets a positional relationship between the position of an opening in the light-blocking member and the focal point of the objective optical system in the sample to an optically conjugate positional relationship, in which in-focus fluorescence emitted from the focal point passes through the light-blocking member
Implementation Method 3
a detector for detecting the fluorescence having passed through the light-blocking member
Implementation Method 4
an objective optical system that focuses the excitation light scanned by the scanner onto a sample and that collects fluorescence generated by the sample
Data Source
AI summary
Provided is a microscope including: a scanner for scanning excitation light; an objective optical system that focuses the excitation light onto a sample and that collects fluorescence generated by the sample; a light-blocking member for transmitting a portion of the collected fluorescence; a detector for detecting the fluorescence; a setting unit that allows types of fluorescence having passed through the light-blocking member to be detected by the detector at different times in two types of positional relationships in which the positional relationship between the position of an opening in the light-blocking member and a focal point of the objective optical system in the sample is set to an optically conjugate positional relationship, in which in-focus fluorescence passes through the light-blocking member, and to an optically nonconjugate positional relationship; and an arithmetic operation unit for calculating the difference between fluorescence signals acquired at different times by the detector.