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Microscopy|44 Article(s)
Advances in Chromatic Confocal Microscopy
Wanyun Ding, Yuhang Wang, Tao Zhang, Hao Qin, and Jixiang Wang
Chromatic confocal microscopy (CCM) combines the high spatial resolution of confocal microscope and the high wavelength resolution of spectral analysis. By virtue of the high precision, strong applicability, and nondestructive detection, it is widely used in the fields of industrial production, biomedicine, semiconductor chips, and other fields. This paper first introduces the principle of point chromatic confocal system and points out its drawback of low detection efficiency. Second, for improving the key performance indexes of chromatic confocal microscopy, the main achievements made in light source, dispersive objective lens, and spectral signal detection are described, and qualitative comparisons are made between various types of light sources. Subsequently, the scanning methods of chromatic confocal microscopy are demonstrated, the relevant research progress is sorted out, and the advantages and disadvantages of the relevant methods are summarized. Finally, the future developments of chromatic confocal microscopy are also prospected. Chromatic confocal microscopy (CCM) combines the high spatial resolution of confocal microscope and the high wavelength resolution of spectral analysis. By virtue of the high precision, strong applicability, and nondestructive detection, it is widely used in the fields of industrial production, biomedicine, semiconductor chips, and other fields. This paper first introduces the principle of point chromatic confocal system and points out its drawback of low detection efficiency. Second, for improving the key performance indexes of chromatic confocal microscopy, the main achievements made in light source, dispersive objective lens, and spectral signal detection are described, and qualitative comparisons are made between various types of light sources. Subsequently, the scanning methods of chromatic confocal microscopy are demonstrated, the relevant research progress is sorted out, and the advantages and disadvantages of the relevant methods are summarized. Finally, the future developments of chromatic confocal microscopy are also prospected.
Laser & Optoelectronics Progress
- Publication Date: Mar. 25, 2024
- Vol. 61, Issue 6, 0618024 (2024)
Research on Snapshot Microspectral Imaging System and Algae Classification Recognition
Dongliang Li, Hongxing Cai, Yu Ren, Shuang Li, Yangyang Hua, Tingting Wang, Jianwei Zhou, Guannan Qu, Shuo Wang, Yangming Cao, and Hengyuan Zhang
Current detection modules in microspectral imaging systems primarily comprise push-broom spectral imagers, which cannot observe dynamic microscopic samples. In this study, leveraging the capabilities of metamaterial broad-spectrum-modulated spectroscopic imaging technology, a snapshot spectroscopic camera was developed and used as a detector module to form a novel snapshot microscopic spectroscopic imaging system with a microscope module. This system enables real-time acquisition of spectral curves and image information from samples. Additionally, we used the developed system to obtain the absorption spectral curves of different algae and further used a image segmentation recognition algorithm based on a support vector machine to recognize dynamic algae samples in water. A total of 80 samples were tested in this experiment, yielding 100% accuracy and 65.52% recall for the prediction results. Thus this result forms the foundation for the application of snapshot spectral imaging technology in the field of microscopy. Current detection modules in microspectral imaging systems primarily comprise push-broom spectral imagers, which cannot observe dynamic microscopic samples. In this study, leveraging the capabilities of metamaterial broad-spectrum-modulated spectroscopic imaging technology, a snapshot spectroscopic camera was developed and used as a detector module to form a novel snapshot microscopic spectroscopic imaging system with a microscope module. This system enables real-time acquisition of spectral curves and image information from samples. Additionally, we used the developed system to obtain the absorption spectral curves of different algae and further used a image segmentation recognition algorithm based on a support vector machine to recognize dynamic algae samples in water. A total of 80 samples were tested in this experiment, yielding 100% accuracy and 65.52% recall for the prediction results. Thus this result forms the foundation for the application of snapshot spectral imaging technology in the field of microscopy.
Laser & Optoelectronics Progress
- Publication Date: Mar. 25, 2024
- Vol. 61, Issue 6, 0618023 (2024)
Multifocus Microscopic Image Fusion Algorithm
Hongyu Fu, Yan Gong, Luhan Wang, Yanwei Zhang, Song Lang, Zhi Zhang, and Hanqing Zheng
In the process of microscopic imaging, the system's depth-of-field limitation results in considerable differences in the focused positions across various planes along the axial direction. This leads to partial overlap of focused regions among microscopic images from different planes. Current multifocus fusion algorithms often struggle to simultaneously extract and merge the sharpest focused parts from multiple microscopic images. Hence, this article proposes a multifocus microscopic image fusion algorithm. First, a Gaussian-like four-neighborhood gradient operator was constructed and combined with fast guided filtering to extract high-frequency focus information. Additionally, a small region focus measurement method was introduced to enhance the extraction for high-frequency focus information from sharply focused regions considering the overlap of focus information and the substantial number of pixels in wide-field microscopic image sequences. This method effectively fuses the best focus points from multiple images. Through experiments, three sets of microscopic multifocus image sequences covering the diagonal fields-of-view of 4 mm and 2 mm were captured and testing was conducted. Through comparative analysis against five commonly used multifocus image fusion algorithms, our algorithm yields an average improvement in the peak signal-to-noise ratio of 2.4772 and surpasses a structural similarity index of 0.9400. These results exhibit superior fusion effects obtained by the proposed algorithm in the focused regions, rendering fused images enriched in details and high clarity. This algorithm meets the accuracy requirements for multifocus image fusion in applications involving large field-of-view microscopic images. In the process of microscopic imaging, the system's depth-of-field limitation results in considerable differences in the focused positions across various planes along the axial direction. This leads to partial overlap of focused regions among microscopic images from different planes. Current multifocus fusion algorithms often struggle to simultaneously extract and merge the sharpest focused parts from multiple microscopic images. Hence, this article proposes a multifocus microscopic image fusion algorithm. First, a Gaussian-like four-neighborhood gradient operator was constructed and combined with fast guided filtering to extract high-frequency focus information. Additionally, a small region focus measurement method was introduced to enhance the extraction for high-frequency focus information from sharply focused regions considering the overlap of focus information and the substantial number of pixels in wide-field microscopic image sequences. This method effectively fuses the best focus points from multiple images. Through experiments, three sets of microscopic multifocus image sequences covering the diagonal fields-of-view of 4 mm and 2 mm were captured and testing was conducted. Through comparative analysis against five commonly used multifocus image fusion algorithms, our algorithm yields an average improvement in the peak signal-to-noise ratio of 2.4772 and surpasses a structural similarity index of 0.9400. These results exhibit superior fusion effects obtained by the proposed algorithm in the focused regions, rendering fused images enriched in details and high clarity. This algorithm meets the accuracy requirements for multifocus image fusion in applications involving large field-of-view microscopic images.
Laser & Optoelectronics Progress
- Publication Date: Mar. 25, 2024
- Vol. 61, Issue 6, 0618022 (2024)
Automatic Exposure Optimization Method for HDMI Digital Microscopy Cameras
Mengwei Zhai, and Feihong Yu
An automatic exposure optimization method for mitigating overexposure, slow convergence speed, and instability in HDMI digital microscopy cameras due to the inconsistent reflection levels of different targets during observation is proposed. Compared with a traditional automatic exposure control method, the proposed method facilitates exposure control based on the region of interest, metering, and brightness statistics on any area in RAW images, thus expanding the dynamic range of brightness statistics and facilitating exposure control for different targets. In addition, the method improves the accuracy of brightness statistics and mitigates overexposure caused by extremely bright or dark targets. A variable exposure adjustment step size is set for the dynamic adjustment of exposure step size according to the current brightness level, achieving coarse and fine adjustment of exposure to balance exposure convergence speed and exposure accuracy. Finally, experiments were performed using actual cameras. The proposed method reduced the exposure deviation by half compared with the traditional automatic exposure method. The exposure convergence time was shortened by more than half, and the fastest exposure convergence was achieved within four frames of an image. An automatic exposure optimization method for mitigating overexposure, slow convergence speed, and instability in HDMI digital microscopy cameras due to the inconsistent reflection levels of different targets during observation is proposed. Compared with a traditional automatic exposure control method, the proposed method facilitates exposure control based on the region of interest, metering, and brightness statistics on any area in RAW images, thus expanding the dynamic range of brightness statistics and facilitating exposure control for different targets. In addition, the method improves the accuracy of brightness statistics and mitigates overexposure caused by extremely bright or dark targets. A variable exposure adjustment step size is set for the dynamic adjustment of exposure step size according to the current brightness level, achieving coarse and fine adjustment of exposure to balance exposure convergence speed and exposure accuracy. Finally, experiments were performed using actual cameras. The proposed method reduced the exposure deviation by half compared with the traditional automatic exposure method. The exposure convergence time was shortened by more than half, and the fastest exposure convergence was achieved within four frames of an image.
Laser & Optoelectronics Progress
- Publication Date: Mar. 25, 2024
- Vol. 61, Issue 6, 0618021 (2024)
Application of Flat-Field Quantitative Phase Microscopy for Observing Mitochondrial Dynamics in Bone Marrow Mesenchymal Stem Cells (Invited)
Taiqiang Dai, Ying Ma, Yuxuan Du, Yan Hou, Lü Qianxin, Juan Kang, Baoli Yao, Peng Gao, and Liang Kong
To investigate the feasibility of label-free imaging using flat-field quantitative phase microscopy for observing mitochondrial dynamics in mesenchymal stem cells (MSCs) derived from bone marrow, SD rat bone marrow MSCs were isolated and cultured. Following passages and purification, they were seeded into confocal culture dishes and placed under a flat-field quantitative phase microscope developed by our team. Label-free observations were conducted over an extended period after confirming mitochondrial characteristics using fluorescence and phase dual channels. The mitochondrial division, fusion processes, and mitochondrial changes during cell apoptosis observed using the flat-field quantitative phase microscope were analyzed. The mitochondria observed via unlabeled flat-field quantitative phase microscopy exhibited complete overlap with fluorescently labeled mitochondria, demonstrating the microscope's capability to visualize mitochondria and conduct unlabeled high-resolution imaging. Moreover, the flat-field quantitative phase microscope facilitated long-term unlabeled observations of bone marrow MSCs under cultivation conditions, enabling high-resolution recording of mitochondrial division and fusion processes. Furthermore, we used the flat-field quantitative phase microscope to record mitochondria changes under CCCP treatment for the first time, visually presenting the apoptosis via mitochondrial pathway. The flat-field quantitative phase microscope allows for prolonged, unlabeled, high-resolution observations of cultured cells, offering a new tool for investigating mitochondrial dynamics. To investigate the feasibility of label-free imaging using flat-field quantitative phase microscopy for observing mitochondrial dynamics in mesenchymal stem cells (MSCs) derived from bone marrow, SD rat bone marrow MSCs were isolated and cultured. Following passages and purification, they were seeded into confocal culture dishes and placed under a flat-field quantitative phase microscope developed by our team. Label-free observations were conducted over an extended period after confirming mitochondrial characteristics using fluorescence and phase dual channels. The mitochondrial division, fusion processes, and mitochondrial changes during cell apoptosis observed using the flat-field quantitative phase microscope were analyzed. The mitochondria observed via unlabeled flat-field quantitative phase microscopy exhibited complete overlap with fluorescently labeled mitochondria, demonstrating the microscope's capability to visualize mitochondria and conduct unlabeled high-resolution imaging. Moreover, the flat-field quantitative phase microscope facilitated long-term unlabeled observations of bone marrow MSCs under cultivation conditions, enabling high-resolution recording of mitochondrial division and fusion processes. Furthermore, we used the flat-field quantitative phase microscope to record mitochondria changes under CCCP treatment for the first time, visually presenting the apoptosis via mitochondrial pathway. The flat-field quantitative phase microscope allows for prolonged, unlabeled, high-resolution observations of cultured cells, offering a new tool for investigating mitochondrial dynamics.
Laser & Optoelectronics Progress
- Publication Date: Mar. 25, 2024
- Vol. 61, Issue 6, 0618020 (2024)
Development and Application of Light Sheet Fluorescence Microscopy Technology (Invited)
Yao Zhou, and Peng Fei
In recent decades, the advent of light sheet fluorescence microscopy as an innovative technique in fluorescence microscopy has significantly enhanced the high spatiotemporal resolution imaging capabilities of tissue and cellular structures and functions in life science research. Compared to traditional epi-fluorescence microscopy techniques, light sheet microscopy illuminates biological samples selectively, greatly improving photon utilization efficiency, reducing phototoxicity, and significantly increasing imaging speed. Since its introduction, light sheet microscopy has gradually expanded its application field in life science research, ranging from embryology and neuroscience to tumor studies, among others. It can not only be used to observe the basic structures of cells and tissues but also for real-time monitoring of dynamic changes in biological processes. Furthermore, its multiscale characteristics make it suitable for observations across multiple scales from macro to micro. This article reviews the applications and developments of light sheet microscopy in high-throughput imaging, high-precision imaging, and usability, aiming to provide life science researchers with comprehensive understanding and reference, and to promote its application and development in more fields. In recent decades, the advent of light sheet fluorescence microscopy as an innovative technique in fluorescence microscopy has significantly enhanced the high spatiotemporal resolution imaging capabilities of tissue and cellular structures and functions in life science research. Compared to traditional epi-fluorescence microscopy techniques, light sheet microscopy illuminates biological samples selectively, greatly improving photon utilization efficiency, reducing phototoxicity, and significantly increasing imaging speed. Since its introduction, light sheet microscopy has gradually expanded its application field in life science research, ranging from embryology and neuroscience to tumor studies, among others. It can not only be used to observe the basic structures of cells and tissues but also for real-time monitoring of dynamic changes in biological processes. Furthermore, its multiscale characteristics make it suitable for observations across multiple scales from macro to micro. This article reviews the applications and developments of light sheet microscopy in high-throughput imaging, high-precision imaging, and usability, aiming to provide life science researchers with comprehensive understanding and reference, and to promote its application and development in more fields.
Laser & Optoelectronics Progress
- Publication Date: Mar. 25, 2024
- Vol. 61, Issue 6, 0618019 (2024)
A New Weapon For Super-Resolution Imaging: Lanthanide Ion Doped Upconversion Nanoparticles (Invited)
Ziqi Li, Xiaolan Zhong, Chaohao Chen, and Fan Wang
Optical super-resolution imaging technology, acknowledged by the Nobel Prize in Chemistry for transcending optical diffraction limits, has revolutionised life science research with its groundbreaking observation scale. However, conventional super-resolution fluorescence microscopes face challenges, requiring intricate optical systems that often result in significant phototoxicity and low temporal resolution, limiting their widespread use in biomedical research. Therefore, research teams actively seek alternative fluorescent probes with near-infrared capabilities, high brightness, and resistance to photobleaching, aiming to extend the application of super-resolution microscopy in biomedical research. Rare-earth nanomaterials, renowned for their exceptional physicochemical properties such as anti-Stokes spectral shift, lack of background noise, resistance to photobleaching, photostability, low toxicity, and high imaging penetration, have emerged as stable and superior inorganic fluorescent probes. This review paper provides a brief overview of the luminescent mechanism of upconversion nanoparticles, exploring the primary constraints in achieving photon upconversion in nanostructured materials. Additionally, it highlights the applications and advantages of lanthanide-doped upconversion nanoparticles in super-resolution biological imaging, molecular detection, and other domains. These advantages encompass reducing laser power requirements, addressing technical challenges in coupling, improving laser scanning imaging resolution and speed, and enhancing multiplexing imaging efficiency. This paper concludes by emphasizing significant challenges in particle synthesis, proposing feasible improvement measures, and outlining prospects for future development. It establishes a robust theoretical foundation and provides technical support for the widespread integration of rare-earth nanomaterials in the field of life imaging sciences. Optical super-resolution imaging technology, acknowledged by the Nobel Prize in Chemistry for transcending optical diffraction limits, has revolutionised life science research with its groundbreaking observation scale. However, conventional super-resolution fluorescence microscopes face challenges, requiring intricate optical systems that often result in significant phototoxicity and low temporal resolution, limiting their widespread use in biomedical research. Therefore, research teams actively seek alternative fluorescent probes with near-infrared capabilities, high brightness, and resistance to photobleaching, aiming to extend the application of super-resolution microscopy in biomedical research. Rare-earth nanomaterials, renowned for their exceptional physicochemical properties such as anti-Stokes spectral shift, lack of background noise, resistance to photobleaching, photostability, low toxicity, and high imaging penetration, have emerged as stable and superior inorganic fluorescent probes. This review paper provides a brief overview of the luminescent mechanism of upconversion nanoparticles, exploring the primary constraints in achieving photon upconversion in nanostructured materials. Additionally, it highlights the applications and advantages of lanthanide-doped upconversion nanoparticles in super-resolution biological imaging, molecular detection, and other domains. These advantages encompass reducing laser power requirements, addressing technical challenges in coupling, improving laser scanning imaging resolution and speed, and enhancing multiplexing imaging efficiency. This paper concludes by emphasizing significant challenges in particle synthesis, proposing feasible improvement measures, and outlining prospects for future development. It establishes a robust theoretical foundation and provides technical support for the widespread integration of rare-earth nanomaterials in the field of life imaging sciences.
Laser & Optoelectronics Progress
- Publication Date: Mar. 25, 2024
- Vol. 61, Issue 6, 0618018 (2024)
New Light in Microscopic Exploration: Portable Photoacoustic Microscopy (Invited)
Mingli Sun, Chiye Li, Ruimin Chen, and Junhui Shi
Photoacoustic imaging (PAI), a biomedical imaging mode that combines the high contrast of optical imaging with the deep penetration of ultrasonic imaging, has developed rapidly in recent years. Among them, photoacoustic microscopy (PAM), as one of the important implementation methods of photoacoustic imaging, can achieve micron-level or even hundreds of nanometer-level resolution on the millimeter imaging depth, and can achieve high-resolution imaging of biological tissue structure, function, and molecules, and has been widely used in clinical diagnosis, skin disease detection, ophthalmology, and other fields. In this paper, the working principle and implementation of PAM are first introduced, and then the research progress of portable PAM technology is reviewed from the handheld and semi-handheld, brain wearable, and integrated multi-mode. Then, the challenges faced by the portable PAM technology are discussed, and finally the prospects are summarized. Photoacoustic imaging (PAI), a biomedical imaging mode that combines the high contrast of optical imaging with the deep penetration of ultrasonic imaging, has developed rapidly in recent years. Among them, photoacoustic microscopy (PAM), as one of the important implementation methods of photoacoustic imaging, can achieve micron-level or even hundreds of nanometer-level resolution on the millimeter imaging depth, and can achieve high-resolution imaging of biological tissue structure, function, and molecules, and has been widely used in clinical diagnosis, skin disease detection, ophthalmology, and other fields. In this paper, the working principle and implementation of PAM are first introduced, and then the research progress of portable PAM technology is reviewed from the handheld and semi-handheld, brain wearable, and integrated multi-mode. Then, the challenges faced by the portable PAM technology are discussed, and finally the prospects are summarized.
Laser & Optoelectronics Progress
- Publication Date: Mar. 25, 2024
- Vol. 61, Issue 6, 0618017 (2024)
Super-Resolution Fluorescence Microscopy for Cilia Investigation and Ciliopathy Diagnosis (Invited)
Zhen Liu, and Yang Wu
The last two decades have witnessed the invention and development of super-resolution microscopy (SRM) that breaks the diffraction limit of light and pushes the fluorescence microscopy resolution to several nanometers. While SRM is widely used in biological studies, such as resolving subdiffraction structures, molecular mapping, tracking single molecules, probing protein-protein interactions, and observing organelle dynamics, its direct application in translational medicine, such as disease diagnosis, is still preliminary. Despite their small size, cilia play a crucial role as organelles in cell signaling and motility, with defects in cilia leading to ciliopathy. Similar to other miniature organelles and macromolecular complexes, cilia are ideal for super-resolution imaging. In this review, we will 1) introduce cilia and ciliopathy, 2) show how SRM extends our knowledge of cilia, and 3) focus on how SRM improves the diagnosis of motile ciliopathies. The last two decades have witnessed the invention and development of super-resolution microscopy (SRM) that breaks the diffraction limit of light and pushes the fluorescence microscopy resolution to several nanometers. While SRM is widely used in biological studies, such as resolving subdiffraction structures, molecular mapping, tracking single molecules, probing protein-protein interactions, and observing organelle dynamics, its direct application in translational medicine, such as disease diagnosis, is still preliminary. Despite their small size, cilia play a crucial role as organelles in cell signaling and motility, with defects in cilia leading to ciliopathy. Similar to other miniature organelles and macromolecular complexes, cilia are ideal for super-resolution imaging. In this review, we will 1) introduce cilia and ciliopathy, 2) show how SRM extends our knowledge of cilia, and 3) focus on how SRM improves the diagnosis of motile ciliopathies.
Laser & Optoelectronics Progress
- Publication Date: Mar. 25, 2024
- Vol. 61, Issue 6, 0618016 (2024)
Three-Dimensional Orientation and Localization Microscopy of Single Molecules Using Super-Resolution Imaging Technology (Invited)
Ruihang Zhao, and Jin Lu
Super-resolution microscopy with nanoscale spatial resolution has become an important imaging tool in life science research. As a super-resolution technique, single-molecule localization microscopy enables us to localize, identify, and study the unique behaviors of single molecules. At the single-molecule level, the emitted fluorescence signal is highly anisotropic. Resolving the polarization or three-dimensional orientation of single fluorescent molecules is an emerging field in super-resolution microscopy. In this review, we describe the three-dimensional orientation of single molecules through super-resolution imaging techniques. These techniques include fluorescence polarization microscopy and single-molecule orientation imaging through point spread function engineering. Furthermore, we discuss other polarization super-resolution imaging approaches for the applications of live cells and single nanoparticle studies. Finally, we discuss the potential challenges and future research needs of single-molecule orientation localization microscopy. These challenges and requirements can provide in-depth insights into future research in life sciences. Super-resolution microscopy with nanoscale spatial resolution has become an important imaging tool in life science research. As a super-resolution technique, single-molecule localization microscopy enables us to localize, identify, and study the unique behaviors of single molecules. At the single-molecule level, the emitted fluorescence signal is highly anisotropic. Resolving the polarization or three-dimensional orientation of single fluorescent molecules is an emerging field in super-resolution microscopy. In this review, we describe the three-dimensional orientation of single molecules through super-resolution imaging techniques. These techniques include fluorescence polarization microscopy and single-molecule orientation imaging through point spread function engineering. Furthermore, we discuss other polarization super-resolution imaging approaches for the applications of live cells and single nanoparticle studies. Finally, we discuss the potential challenges and future research needs of single-molecule orientation localization microscopy. These challenges and requirements can provide in-depth insights into future research in life sciences.
Laser & Optoelectronics Progress
- Publication Date: Mar. 25, 2024
- Vol. 61, Issue 6, 0618015 (2024)
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