Confocal Microscope Pinhole Array Resolution Enhancement
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Solution Overview
Problem
Current confocal microscopy techniques face limitations in increasing resolution due to diffraction constraints and require complex methods involving fluorescent labels or specific excitation conditions, leading to technical complexity and high costs.
Innovation Solution
A process involving two pinholes of different sizes, where a basic light beam passes through one or more first pinholes and then second pinholes of larger size, with images processed using Fourier transforms and deconvolution to estimate and correct the point spread function, allowing for increased resolution without requiring auxiliary parameters or interpolation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If structured illumination microscopy is used to surpass the lateral resolution limit, then resolution is improved by a factor of two, but device complexity and manufacturing costs increase
Solution Approach 1:
The invention segments the pinhole array into multiple groups, where each group contains pinholes of different sizes. By selectively activating different groups during scanning, the system achieves super-resolution without requiring complex structured illumination optics. The segmentation of pinhole sizes enables differential sampling of the point spread function, facilitating resolution enhancement through computational processing rather than complex optical arrangements.
2Object-affected harmful factors
If pinhole distance is increased to prevent pinhole cross-talk, then background noise is reduced, but signal intensity and signal-to-noise ratio decrease
Solution Approach 1:
The invention applies local quality by assigning different pinhole sizes to different spatial locations within the pinhole array. Each pinhole's size is optimized for its specific position and function. Smaller pinholes provide better spatial resolution with less cross-talk, while larger pinholes collect more signal. This localized optimization of pinhole characteristics allows the system to maintain appropriate signal-to-noise ratios across different regions of the sample without requiring uniform increases in pinhole separation.
3Measurement precision
If super-resolution methods using fluorescent labels are used, then resolution beyond diffraction limit is achieved, but the method requires specific fluorescent probes and excitation conditions
Solution Approach 1:
The invention achieves universal applicability by using a pinhole array with multiple size groups that can process images from any fluorescent sample type without requiring specific fluorescent labels or specialized excitation conditions. The differential sampling approach works with any fluorescence emission, making the super-resolution capability applicable to diverse samples including live cells, fixed tissues, and various fluorescent proteins without needing to optimize for specific fluorophore properties.
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 enhances image resolution while maintaining signal quality and allowing use with any light frequency or modulation, acting on any sample without specific fluorescent requirements, without substantial mechanical modifications to the microscope.
Implementation Method 1
calculating an estimate g of the PSF of the optics of the microscope, through an inverse Fourier transform of the Fourier transform ĝ calculated as follows
Implementation Method 2
deconvolving the acquired second overall image A k using the estimate g of the PSF of the optics of the microscope through a main deconvolution algorithm
Data Source
Figure 1
Figure 2~3
Figure 4
AI summary
Process of acquiring and processing images in confocal microscopy, comprising the following steps: A. acquiring (500, 510; 520, 530) a first overall image Ak-1 of a plane of a sample (160) scanning the same with a first excitation light beam, obtained by making a basic light beam pass through first pinholes (1255; 1255') and transmitted to an optics (155) of a confocal microscope (100) that is focused on the plane of the sample (160), and acquiring a second overall image Ak of the plane of the sample (160) scanning the same with a second excitation light beam, obtained by making said basic light beam pass through second pinholes (1265; 1265') having same shape and size (L2; La2, Lb2) larger than the first pinholes (1255; 1255'); B. calculating (540) an estimate g of the PSF of the optics (155) of the microscope (100), through an inverse Fourier transform of formula (I): where Âk -1(ω) is the Fourier transform of the acquired first overall image Ak-1 and Âk(ω)is the Fourier transform of the acquired second overall image Ak; and C. deconvolving (550) the acquired second overall image Ak using the estimate g of the PSF of the optics (155) of the microscope (100) through a main deconvolution algorithm, obtaining the resulting image / of the entire plane of the sample (160): I = Ak• g.