An Auto-Focus Method of Microscope for the Surface Structure of Transparent Materials under Transmission Illumination

Liao, Yang; Xiong, Yan; Yang, Yunhong

doi:10.3390/s21072487

Cited by 4 publications

(1 citation statement)

References 28 publications

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“…However, to acquire high quality and crispy clear microscope images, other than a good quality microscope optical system itself, the key is to let the microscope to work under its best focusing state. There are still a lot of research works being report in this field [1][2][3][4]. In many applications, the surface under observation may constantly varying its relative distance to the optical microscope, therefore requiring adjustment of focusing distance to maintain best focus.…”

Section: Introductionmentioning

confidence: 99%

Study of Microscope Autofocus Based on Weighted Light Intensity

Lu¹,

Tu²,

Wu³

et al. 2021

AJPA

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Microscopes auto focusing is critically important for a great spectrum of scientific and industrial applications.Finding the best focus position for variety of surfaces under measurement proved to be challenging. In order to provide a rapid and accurate method for microscope focusing positioning, a laser focus deviate system was employed for microscope focusing position. The relationship between focusing sensitivity and the surface under measurement was investigated. First, the laser focus deviate technique is introduced in light of characteristic parameters such as the diameter of the focused spot, the laser wavelength and the distance from the optimal focus. Then, the focusing displacement versus spot position curve fitting is performed for a smooth isotropic surface to directly locate the best focusing position. Following, it is analyzed that when applied to anisotropic and rough surfaces, the centroid of the image spot after reflection/scattering from the surface will deviate from its original light intensity distribution, thus deviate from the calculated optimal focus position and cause focusing errors. Finally, a criterion of weighted light intensity value Q is introduced to achieve fast focusing of anisotropic surfaces in combination with the laser focusing deviate technique. The experimental results show that for a 20× microscope objective system, the focusing accuracy of this focusing positioning method is 1.7 µm on smooth surfaces; and is at the micro meter level for rough surfaces. It basically meets the requirements for rapid, stable and reliable microscope auto focusing needs.

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Section: Introductionmentioning

confidence: 99%

Study of Microscope Autofocus Based on Weighted Light Intensity

Lu¹,

Tu²,

Wu³

et al. 2021

AJPA

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Maximum Gradient Autofocus Technology of Microsporidia Images Based on Color Feature

Xiong

Bai³

et al. 2023

Int. J. Patt. Recogn. Artif. Intell.

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There are many impurities in the microscopic images of extracted microsporidia samples of Bombyx mori pebrine, and Bombyx mori pebrine with elliptical symmetric shape has certain fluidity and obvious stratification. Traditional focusing methods cannot accurately locate the main regions of microsporidia images, and the focusing effect is poor. On this basis, an automatic focusing method combining the microsporidia image features and the evaluation and determination of maximum gradient direction is proposed. First, the HSV color space with stable color information is used to extract the suspected positions of microsporidia targets, so that the interference of some impurities under complex backgrounds is removed and the redundancy of image content calculation is reduced. Then, combined with the light green features of Bombyx mori pebrine, the G-component gray image of microsporidia in the RGB color space is used to extract the significant gradient region. A dynamic focus window is constructed to accurately locate the target region and reduce the influence of microsporidia flow on the focus evaluation function and the bimodal interference caused by impurities. Finally, the maximum second-order difference is obtained through the four-dimensional gradient distribution, and the focus sharpness evaluation function is formulated to adapt to the microsporidia shape and improve the sensitivity of the focus function. The experiments show that under the dynamic window of microsporidia color gradient of different samples, the sharpness ratio and the highest sensitivity factor of the focus evaluation function proposed in this paper can reach 0.06341 and 0.95, respectively. It can meet the accurate and sensitive autofocus of microscopic images of color microsporidia samples under complex backgrounds.

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Illumination angle correction during image acquisition in light-sheet fluorescence microscopy using deep learning

Ratnam

Ghashghaei

et al. 2022

Biomed. Opt. Express

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Light-sheet fluorescence microscopy (LSFM) is a high-speed imaging technique that provides optical sectioning with reduced photodamage. LSFM is routinely used in life sciences for live cell imaging and for capturing large volumes of cleared tissues. LSFM has a unique configuration, in which the illumination and detection paths are separated and perpendicular to each other. As such, the image quality, especially at high resolution, largely depends on the degree of overlap between the detection focal plane and the illuminating beam. However, spatial heterogeneity within the sample, curved specimen boundaries, and mismatch of refractive index between tissues and immersion media can refract the well-aligned illumination beam. This refraction can cause extensive blur and non-uniform image quality over the imaged field-of-view. To address these issues, we tested a deep learning-based approach to estimate the angular error of the illumination beam relative to the detection focal plane. The illumination beam was then corrected using a pair of galvo scanners, and the correction significantly improved the image quality across the entire field-of-view. The angular estimation was based on calculating the defocus level on a pixel level within the image using two defocused images. Overall, our study provides a framework that can correct the angle of the light-sheet and improve the overall image quality in high-resolution LSFM 3D image acquisition.

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An Auto-Focus Method of Microscope for the Surface Structure of Transparent Materials under Transmission Illumination

Cited by 4 publications

References 28 publications

Study of Microscope Autofocus Based on Weighted Light Intensity

Study of Microscope Autofocus Based on Weighted Light Intensity

Maximum Gradient Autofocus Technology of Microsporidia Images Based on Color Feature

Illumination angle correction during image acquisition in light-sheet fluorescence microscopy using deep learning

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