2,070 results on '"Optical sectioning"'
Search Results
2. In Vivo Intelligent Fluorescence Endo‐Microscopy by Varifocal Meta‐Device and Deep Learning.
- Author
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Chia, Yu‐Hsin, Liao, Wei‐Hao, Vyas, Sunil, Chu, Cheng Hung, Yamaguchi, Takeshi, Liu, Xiaoyuan, Tanaka, Takuo, Huang, Yi‐You, Chen, Mu Ku, Chen, Wen‐Shiang, Tsai, Din Ping, and Luo, Yuan
- Subjects
- *
DEEP learning , *FOCAL length , *FLUORESCENCE , *OPTICAL devices , *TIME complexity - Abstract
Endo‐microscopy is crucial for real‐time 3D visualization of internal tissues and subcellular structures. Conventional methods rely on axial movement of optical components for precise focus adjustment, limiting miniaturization and complicating procedures. Meta‐device, composed of artificial nanostructures, is an emerging optical flat device that can freely manipulate the phase and amplitude of light. Here, an intelligent fluorescence endo‐microscope is developed based on varifocal meta‐lens and deep learning (DL). The breakthrough enables in vivo 3D imaging of mouse brains, where varifocal meta‐lens focal length adjusts through relative rotation angle. The system offers key advantages such as invariant magnification, a large field‐of‐view, and optical sectioning at a maximum focal length tuning range of ≈2 mm with 3 µm lateral resolution. Using a DL network, image acquisition time and system complexity are significantly reduced, and in vivo high‐resolution brain images of detailed vessels and surrounding perivascular space are clearly observed within 0.1 s (≈50 times faster). The approach will benefit various surgical procedures, such as gastrointestinal biopsies, neural imaging, brain surgery, etc. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
3. High Fidelity Full-Color Optical Sectioning Structured Illumination Microscopy by Fourier Domain Based Reconstruction.
- Author
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Dang, Shipei, Qian, Jia, Ma, Wang, Ma, Rui, Li, Xing, Wang, Siying, Bai, Chen, Dan, Dan, and Yao, Baoli
- Subjects
COLOR space ,MICROSCOPY ,BIOLOGICAL specimens ,POLLEN ,PHYSIOLOGICAL adaptation - Abstract
The natural color of biological specimens plays a crucial role in body protection, signaling, physiological adaptations, etc. Full-color optical sectioning structured illumination microscopy (OS-SIM) color is a promising approach that can reconstruct biological specimens in three-dimension meanwhile maintaining their natural color. Full-color OS-SIM takes the advantages of rapid imaging speed, compatibility with fluorescence and non-fluorescence samples, compact configuration, and low cost. However, the commonly used HSV-RMS reconstruction algorithm for full-color OS-SIM faces two issues to be improved. One is the RMS (root-mean-square) OS reconstruction algorithm is prone to background noise, and the other is the reconstruction is bound in RGB and HSV color spaces, consuming more reconstructing time. In this paper, we propose a full-color Fourier-OS-SIM method that allows for the OS reconstruction using the high-frequency spectrum of the sample and thus is immune to the low-frequency background noise. The full-color Fourier-OS-SIM directly runs in the RGB color space, providing an easy way to restore the color information. Simulation and experiments with various samples (pollen grains and tiny animals) demonstrate that the full-color Fourier-OS-SIM method is superior to the HSV-RMS method regarding background noise suppression. Moreover, benefiting from the background noise suppression merit, the quantitative morphological height map analysis with the full-color Fourier-OS-SIM method is more accurate. The proposed full-color Fourier-OS-SIM method is expected to find broad applications in biological and industrial fields where the 3D morphology and the color information of objects both need to be recovered. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
4. In Vivo Intelligent Fluorescence Endo‐Microscopy by Varifocal Meta‐Device and Deep Learning
- Author
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Yu‐Hsin Chia, Wei‐Hao Liao, Sunil Vyas, Cheng Hung Chu, Takeshi Yamaguchi, Xiaoyuan Liu, Takuo Tanaka, Yi‐You Huang, Mu Ku Chen, Wen‐Shiang Chen, Din Ping Tsai, and Yuan Luo
- Subjects
deep learning ,endoscopy ,HiLo fluorescence imaging ,metalens ,optical sectioning ,telecentric configuration ,Science - Abstract
Abstract Endo‐microscopy is crucial for real‐time 3D visualization of internal tissues and subcellular structures. Conventional methods rely on axial movement of optical components for precise focus adjustment, limiting miniaturization and complicating procedures. Meta‐device, composed of artificial nanostructures, is an emerging optical flat device that can freely manipulate the phase and amplitude of light. Here, an intelligent fluorescence endo‐microscope is developed based on varifocal meta‐lens and deep learning (DL). The breakthrough enables in vivo 3D imaging of mouse brains, where varifocal meta‐lens focal length adjusts through relative rotation angle. The system offers key advantages such as invariant magnification, a large field‐of‐view, and optical sectioning at a maximum focal length tuning range of ≈2 mm with 3 µm lateral resolution. Using a DL network, image acquisition time and system complexity are significantly reduced, and in vivo high‐resolution brain images of detailed vessels and surrounding perivascular space are clearly observed within 0.1 s (≈50 times faster). The approach will benefit various surgical procedures, such as gastrointestinal biopsies, neural imaging, brain surgery, etc.
- Published
- 2024
- Full Text
- View/download PDF
5. Whole-brain Optical Imaging: A Powerful Tool for Precise Brain Mapping at the Mesoscopic Level.
- Author
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Jiang, Tao, Gong, Hui, and Yuan, Jing
- Abstract
The mammalian brain is a highly complex network that consists of millions to billions of densely-interconnected neurons. Precise dissection of neural circuits at the mesoscopic level can provide important structural information for understanding the brain. Optical approaches can achieve submicron lateral resolution and achieve "optical sectioning" by a variety of means, which has the natural advantage of allowing the observation of neural circuits at the mesoscopic level. Automated whole-brain optical imaging methods based on tissue clearing or histological sectioning surpass the limitation of optical imaging depth in biological tissues and can provide delicate structural information in a large volume of tissues. Combined with various fluorescent labeling techniques, whole-brain optical imaging methods have shown great potential in the brain-wide quantitative profiling of cells, circuits, and blood vessels. In this review, we summarize the principles and implementations of various whole-brain optical imaging methods and provide some concepts regarding their future development. [ABSTRACT FROM AUTHOR]
- Published
- 2023
- Full Text
- View/download PDF
6. Introduction to Fluorescence Microscopy
- Author
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Macháň, Radek, Pedras, Bruno, Series Editor, Šachl, Radek, editor, and Amaro, Mariana, editor
- Published
- 2023
- Full Text
- View/download PDF
7. Minimal resin embedding of SBF-SEM samples reduces charging and facilitates finding a surface-linked region of interest
- Author
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Barbora Konopová and Jiří Týč
- Subjects
3D imaging ,Arthropod ,High resolution ,Optical sectioning ,ROI localization ,Serial block face ,Zoology ,QL1-991 - Abstract
Abstract Background For decoding the mechanism of how cells and organs function information on their ultrastructure is essential. High-resolution 3D imaging has revolutionized morphology. Serial block face scanning electron microscopy (SBF-SEM) offers non-laborious, automated imaging in 3D of up to ~ 1 mm3 large biological objects at nanometer-scale resolution. For many samples there are obstacles. Quality imaging is often hampered by charging effects, which originate in the nonconductive resin used for embedding. Especially, if the imaged region of interest (ROI) includes the surface of the sample and neighbours the empty resin, which insulates the object. This extra resin also obscures the sample’s morphology, thus making navigation to the ROI difficult. Results Using the example of small arthropods and a fish roe we describe a workflow to prepare samples for SBF-SEM using the minimal resin (MR) embedding method. We show that for imaging of surface structures this simple approach conveniently tackles and solves both of the two major problems—charging and ROI localization—that complicate imaging of SBF-SEM samples embedded in an excess of overlying resin. As the surface ROI is not masked by the resin, samples can be precisely trimmed before they are placed into the imaging chamber. The initial approaching step is fast and easy. No extra trimming inside the microscope is necessary. Importantly, charging is absent or greatly reduced meaning that imaging can be accomplished under good vacuum conditions, typically at the optimal high vacuum. This leads to better resolution, better signal to noise ratio, and faster image acquisition. Conclusions In MR embedded samples charging is minimized and ROI easily targeted. MR embedding does not require any special equipment or skills. It saves effort, microscope time and eventually leads to high quality data. Studies on surface-linked ROIs, or any samples normally surrounded by the excess of resin, would benefit from adopting the technique.
- Published
- 2023
- Full Text
- View/download PDF
8. High Fidelity Full-Color Optical Sectioning Structured Illumination Microscopy by Fourier Domain Based Reconstruction
- Author
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Shipei Dang, Jia Qian, Wang Ma, Rui Ma, Xing Li, Siying Wang, Chen Bai, Dan Dan, and Baoli Yao
- Subjects
structured illumination microscopy ,optical sectioning ,full-color ,3D reconstruction ,Applied optics. Photonics ,TA1501-1820 - Abstract
The natural color of biological specimens plays a crucial role in body protection, signaling, physiological adaptations, etc. Full-color optical sectioning structured illumination microscopy (OS-SIM) color is a promising approach that can reconstruct biological specimens in three-dimension meanwhile maintaining their natural color. Full-color OS-SIM takes the advantages of rapid imaging speed, compatibility with fluorescence and non-fluorescence samples, compact configuration, and low cost. However, the commonly used HSV-RMS reconstruction algorithm for full-color OS-SIM faces two issues to be improved. One is the RMS (root-mean-square) OS reconstruction algorithm is prone to background noise, and the other is the reconstruction is bound in RGB and HSV color spaces, consuming more reconstructing time. In this paper, we propose a full-color Fourier-OS-SIM method that allows for the OS reconstruction using the high-frequency spectrum of the sample and thus is immune to the low-frequency background noise. The full-color Fourier-OS-SIM directly runs in the RGB color space, providing an easy way to restore the color information. Simulation and experiments with various samples (pollen grains and tiny animals) demonstrate that the full-color Fourier-OS-SIM method is superior to the HSV-RMS method regarding background noise suppression. Moreover, benefiting from the background noise suppression merit, the quantitative morphological height map analysis with the full-color Fourier-OS-SIM method is more accurate. The proposed full-color Fourier-OS-SIM method is expected to find broad applications in biological and industrial fields where the 3D morphology and the color information of objects both need to be recovered.
- Published
- 2024
- Full Text
- View/download PDF
9. Bessel Beams in Ophthalmology: A Review.
- Author
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Suchand Sandeep, C. S., Khairyanto, Ahmad, Aung, Tin, and Vadakke Matham, Murukeshan
- Subjects
VECTOR beams ,BESSEL beams ,IMAGING systems ,OPHTHALMOLOGY ,SIGNAL-to-noise ratio - Abstract
The achievable resolution of a conventional imaging system is inevitably limited due to diffraction. Dealing with precise imaging in scattering media, such as in the case of biomedical imaging, is even more difficult owing to the weak signal-to-noise ratios. Recent developments in non-diffractive beams such as Bessel beams, Airy beams, vortex beams, and Mathieu beams have paved the way to tackle some of these challenges. This review specifically focuses on non-diffractive Bessel beams for ophthalmological applications. The theoretical foundation of the non-diffractive Bessel beam is discussed first followed by a review of various ophthalmological applications utilizing Bessel beams. The advantages and disadvantages of these techniques in comparison to those of existing state-of-the-art ophthalmological systems are discussed. The review concludes with an overview of the current developments and the future perspectives of non-diffractive beams in ophthalmology. [ABSTRACT FROM AUTHOR]
- Published
- 2023
- Full Text
- View/download PDF
10. Minimal resin embedding of SBF-SEM samples reduces charging and facilitates finding a surface-linked region of interest.
- Author
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Konopová, Barbora and Týč, Jiří
- Subjects
- *
FISH eggs , *SIGNAL-to-noise ratio , *THREE-dimensional imaging , *SCANNING electron microscopy , *SURFACE structure , *MINIMAL surfaces - Abstract
Background: For decoding the mechanism of how cells and organs function information on their ultrastructure is essential. High-resolution 3D imaging has revolutionized morphology. Serial block face scanning electron microscopy (SBF-SEM) offers non-laborious, automated imaging in 3D of up to ~ 1 mm3 large biological objects at nanometer-scale resolution. For many samples there are obstacles. Quality imaging is often hampered by charging effects, which originate in the nonconductive resin used for embedding. Especially, if the imaged region of interest (ROI) includes the surface of the sample and neighbours the empty resin, which insulates the object. This extra resin also obscures the sample's morphology, thus making navigation to the ROI difficult. Results: Using the example of small arthropods and a fish roe we describe a workflow to prepare samples for SBF-SEM using the minimal resin (MR) embedding method. We show that for imaging of surface structures this simple approach conveniently tackles and solves both of the two major problems—charging and ROI localization—that complicate imaging of SBF-SEM samples embedded in an excess of overlying resin. As the surface ROI is not masked by the resin, samples can be precisely trimmed before they are placed into the imaging chamber. The initial approaching step is fast and easy. No extra trimming inside the microscope is necessary. Importantly, charging is absent or greatly reduced meaning that imaging can be accomplished under good vacuum conditions, typically at the optimal high vacuum. This leads to better resolution, better signal to noise ratio, and faster image acquisition. Conclusions: In MR embedded samples charging is minimized and ROI easily targeted. MR embedding does not require any special equipment or skills. It saves effort, microscope time and eventually leads to high quality data. Studies on surface-linked ROIs, or any samples normally surrounded by the excess of resin, would benefit from adopting the technique. [ABSTRACT FROM AUTHOR]
- Published
- 2023
- Full Text
- View/download PDF
11. Axial Resolution Enhancement of Optical Sectioning Structured Illumination Microscopy Based on Three-Beam Interference.
- Author
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Xiao, Chao, Li, Xing, Qian, Jia, Ma, Wang, Min, Junwei, Gao, Peng, Dan, Dan, and Yao, Baoli
- Subjects
INTERFERENCE microscopy ,OPTICAL resolution ,MICROSCOPY ,THREE-dimensional imaging ,DIFFRACTION patterns ,LIGHTING - Abstract
As a branch of 3D microscopy, optical sectioning structured illumination microscopy (OS-SIM) has the advantages of fast imaging speed, weak photobleaching and phototoxicity, and flexible and compatible configuration. Although the method of using the one-dimensional periodic fringe pattern projected on the sample can remove the out-of-focus background from the in-focus signal, the axial resolution of the final reconstructed 3D image is not improved. Here, we propose a three-beam interference OS-SIM, namely TBOS, instead of the common-used dual-beam interference OS-SIM (DBOS). The three-beam interference scheme has been adopted in 3D super-resolution SIM (3D-SR-SIM), where the fringe phase shifting needs to be along each of the three orientations. In contrast, TBOS applies phase shifting only in one arbitrary direction. We built a TBOS SIM microscope and performed the 3D imaging experiments with 46 nm diameter fluorescent microspheres and a mouse kidney section. The axial resolution of the 3D image obtained with TBOS was enhanced by a factor of 1.36 compared to the DBOS method, consistent with the theoretical analysis and simulation. The OS-SIM with enhanced axial resolution for 3D imaging may find a wide range of applications in the biomedical field. [ABSTRACT FROM AUTHOR]
- Published
- 2023
- Full Text
- View/download PDF
12. Confocal Microscopy
- Author
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Sanderson, Jeremy and Nechyporuk-Zloy, Volodymyr, editor
- Published
- 2022
- Full Text
- View/download PDF
13. Using saturated absorption for superresolution laser scanning transmission microscopy.
- Author
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Nishida, Kentaro, Sato, Hikaru, Oketani, Ryosuke, Mochizuki, Kentaro, Temma, Kenta, Kumamoto, Yasuaki, Tanaka, Hideo, and Fujita, Katsumasa
- Subjects
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MICROSCOPY , *ABSORPTION , *LASERS , *NUMERICAL calculations , *SPATIAL resolution , *HIGH-intensity focused ultrasound - Abstract
We improved the three‐dimensional spatial resolution of laser scanning transmission microscopy by exploiting the saturated absorption of dye molecules. The saturated absorption is induced by the high‐intensity light irradiation and localises the signal within the centre of the focal spot. Our numerical calculation indicates that the spatial resolution in transmission imaging is significantly improved for both lateral and axial directions using nonlinear transmitted signals induced by saturated absorption. We experimentally demonstrated the improvement of the three‐dimensional resolution by observing fine structures of stained rat kidney tissues, which were not able to be visualised by conventional laser scanning transmission microscopy. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
14. Quantitative phase imaging of opaque specimens with flexible endoscopic microscopy.
- Author
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Wang, Jingyi, You, Wu, Jiao, Yuheng, Zhu, Yanhong, Liu, Xiaojun, Jiang, Xiangqian, Hu, Chenfei, and Lu, Wenlong
- Subjects
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MICROSCOPY , *CELL imaging , *WOUND healing , *SPECKLE interferometry , *ENDOMETRIAL diseases , *WAVEFRONTS (Optics) , *ENDOSCOPIC ultrasonography - Abstract
• Our work presents a minimally invasive and portable tool for quantitative phase imaging (QPI) of unlabeled thick samples, with potential applications in biomedical imaging at cellular level resolution. We highlight the features of the system performance and performed experiments and analysis in vivo. • In brief, our fiber-bundle-based endoscopic microscopy (FEM) has a flexible front probe with a small form factor, with a QPI module located at the proximal end. The optical phase transmits through the sample is easily obtained via wavefront modulation and straightforward post-processing. FEM overcomes limitations in competing methods, such as iterative phase calculation, image speckle, intensive modeling of the system transfer function, complicated system setup with lateral scanning module or reference arm, and time cost on sample staining. • We explored biomedical imaging potential of FEM by histological slices and in vivo experiments. For cancer diagnosis, FEM distinguishes benign and diseased endometrial slides with differences on morphology and scattering parameters. For thick tissue, FEM images unlabeled mammalian skin in vivo, identifying the boundary and morphology differences between the scar and normal skin, as well as the variations from the scab to scar during 15 days post wounding, such as fibroblast proliferation and inflammation resolution. These findings contribute significantly to our understanding of wound healing mechanisms, wound care, and the development of therapeutic drugs. • With the quantitative information, high sensitivity, and label-free imaging capabilities, we believe the system can greatly benefit the biomedical community. We hope that it will be of interest to the readers of optics and lasers in engineering and we thank you for considering our manuscript. The flexible endoscope is a minimally invasive tool in clinical settings. Conventional modalities record bright field images that are usually low contrast and report qualitative information of the sample surface. Here, we demonstrated a flexible endoscopic microscopy (FEM) for quantitative phase imaging of unlabeled thick samples. FEM employs a 2.5 mm diameter fiber tip, followed by a wavefront shaping module for direct phase measurement. Our instrument yields lateral and axial resolutions of 1.2 and 8.3 um, respectively. Demonstrating the performance on pathologic slices, thick opaque mammalian wound healing skin in vivo, FEM identifies normal and tumor glandular structures, secreta, and tomographic skin layers. With the advantages of direct phase measurement, cellular level resolution, thin fiber tip, and simple setup, FEM has potential value for investigation in vivo. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
15. Bessel Beams in Ophthalmology: A Review
- Author
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C. S. Suchand Sandeep, Ahmad Khairyanto, Tin Aung, and Murukeshan Vadakke Matham
- Subjects
Bessel beam ,non-diffractive beams ,self-reconstruction ,high-resolution imaging ,ophthalmic imaging ,optical sectioning ,Mechanical engineering and machinery ,TJ1-1570 - Abstract
The achievable resolution of a conventional imaging system is inevitably limited due to diffraction. Dealing with precise imaging in scattering media, such as in the case of biomedical imaging, is even more difficult owing to the weak signal-to-noise ratios. Recent developments in non-diffractive beams such as Bessel beams, Airy beams, vortex beams, and Mathieu beams have paved the way to tackle some of these challenges. This review specifically focuses on non-diffractive Bessel beams for ophthalmological applications. The theoretical foundation of the non-diffractive Bessel beam is discussed first followed by a review of various ophthalmological applications utilizing Bessel beams. The advantages and disadvantages of these techniques in comparison to those of existing state-of-the-art ophthalmological systems are discussed. The review concludes with an overview of the current developments and the future perspectives of non-diffractive beams in ophthalmology.
- Published
- 2023
- Full Text
- View/download PDF
16. Three-dimensional natural color imaging based on focus level correlation algorithm using structured illumination microscopy
- Author
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Mengrui Wang, Tianyu Zhao, Zhaojun Wang, Kun Feng, Jingrong Ren, Yansheng Liang, Shaowei Wang, and Ming Lei
- Subjects
structured illumination microscopy ,optical sectioning ,natural color ,3D image ,focus level correlation algorithm ,Physics ,QC1-999 - Abstract
Taking advantages of high-resolution, natural color restoration, and high imaging speed, optical sectioning structured illumination microscopy (OS-SIM) plays an important role in geology, biology, and material science. However, when encountering chromatic aberration or dealing with samples with semitransparent surface, the HSV (Hue, Saturation, and Value) decoding algorithm suffers intensity deviation and fading color. In this paper, we propose a focus level correlation algorithm for 3D color image reconstruction in OS-SIM. Simulations and experiments demonstrate that the algorithm can restore color of sample authentically, and improve the image processing speed by about 45%. This new algorithm successfully improves the results and the speed of optical sectioning reconstruction, expanding the application of OS-SIM.
- Published
- 2022
- Full Text
- View/download PDF
17. A phasor‐based approach to improve optical sectioning in any confocal microscope with a tunable pinhole.
- Author
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D'Amico, Morgana, Di Franco, Elisabetta, Cerutti, Elena, Barresi, Vincenza, Condorelli, Daniele, Diaspro, Alberto, and Lanzanò, Luca
- Abstract
Confocal fluorescence microscopy is a well‐established imaging technique capable of generating thin optical sections of biological specimens. Optical sectioning in confocal microscopy is mainly determined by the size of the pinhole, a small aperture placed in front of a point detector. In principle, imaging with a closed pinhole provides the highest degree of optical sectioning. In practice, the dramatic reduction of signal‐to‐noise ratio (SNR) at smaller pinhole sizes makes challenging the use of pinhole sizes significantly smaller than 1 Airy Unit (AU). Here, we introduce a simple method to "virtually" perform confocal imaging at smaller pinhole sizes without the dramatic reduction of SNR. The method is based on the sequential acquisition of multiple confocal images acquired at different pinhole aperture sizes and image processing based on a phasor analysis. The implementation is conceptually similar to separation of photons by lifetime tuning (SPLIT), a technique that exploits the phasor analysis to achieve super‐resolution, and for this reason we call this method SPLIT‐pinhole (SPLIT‐PIN). We show with simulated data that the SPLIT‐PIN image can provide improved optical sectioning (i.e., virtually smaller pinhole size) but better SNR with respect to an image obtained with closed pinhole. For instance, two images acquired at 2 and 1 AU can be combined to obtain a SPLIT‐PIN image with a virtual pinhole size of 0.2 AU but with better SNR. As an example of application to biological imaging, we show that SPLIT‐PIN improves confocal imaging of the apical membrane in an in vitro model of the intestinal epithelium. Research Highlights: We describe a method to boost the optical sectioning power of any confocal microscope. The method is based on the sequential acquisition of multiple confocal images acquired at different pinhole aperture sizes. The resulting image series is analyzed using the phasor‐based separation of photons by lifetime tuning (SPLIT) algorithm. The SPLIT‐pinhole (SPLIT‐PIN) method produces images with improved optical sectioning but preserved SNR. This is the first time that the phasor analysis and SPLIT algorithms are used to exploit the spatial information encoded in a tunable pinhole size and to improve optical sectioning of the confocal microscope. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
18. Axial Resolution Enhancement of Optical Sectioning Structured Illumination Microscopy Based on Three-Beam Interference
- Author
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Chao Xiao, Xing Li, Jia Qian, Wang Ma, Junwei Min, Peng Gao, Dan Dan, and Baoli Yao
- Subjects
structured illumination microscopy ,optical sectioning ,three-beam interference ,axial resolution ,3D microscopy ,Applied optics. Photonics ,TA1501-1820 - Abstract
As a branch of 3D microscopy, optical sectioning structured illumination microscopy (OS-SIM) has the advantages of fast imaging speed, weak photobleaching and phototoxicity, and flexible and compatible configuration. Although the method of using the one-dimensional periodic fringe pattern projected on the sample can remove the out-of-focus background from the in-focus signal, the axial resolution of the final reconstructed 3D image is not improved. Here, we propose a three-beam interference OS-SIM, namely TBOS, instead of the common-used dual-beam interference OS-SIM (DBOS). The three-beam interference scheme has been adopted in 3D super-resolution SIM (3D-SR-SIM), where the fringe phase shifting needs to be along each of the three orientations. In contrast, TBOS applies phase shifting only in one arbitrary direction. We built a TBOS SIM microscope and performed the 3D imaging experiments with 46 nm diameter fluorescent microspheres and a mouse kidney section. The axial resolution of the 3D image obtained with TBOS was enhanced by a factor of 1.36 compared to the DBOS method, consistent with the theoretical analysis and simulation. The OS-SIM with enhanced axial resolution for 3D imaging may find a wide range of applications in the biomedical field.
- Published
- 2023
- Full Text
- View/download PDF
19. Design and Development of a Bimodal Optical Instrument for Simultaneous Vibrational Spectroscopy Measurements.
- Author
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Arévalo, Laura A., O'Brien, Stephen A., Lopez, Eneko, Singh, Gajendra Pratap, and Seifert, Andreas
- Subjects
- *
OPTICAL instruments , *SPECTROMETRY , *RAMAN spectroscopy , *INFRARED spectroscopy , *INFRARED spectra , *INFRARED imaging - Abstract
Vibrational spectroscopy techniques are widely used in analytical chemistry, physics and biology. The most prominent techniques are Raman and Fourier-transform infrared spectroscopy (FTIR). Combining both techniques delivers complementary information of the test sample. We present the design, construction, and calibration of a novel bimodal spectroscopy system featuring both Raman and infrared measurements simultaneously on the same sample without mutual interference. The optomechanical design provides a modular flexible system for solid and liquid samples and different configurations for Raman. As a novel feature, the Raman module can be operated off-axis for optical sectioning. The calibrated system demonstrates high sensitivity, precision, and resolution for simultaneous operation of both techniques and shows excellent calibration curves with coefficients of determination greater than 0.96. We demonstrate the ability to simultaneously measure Raman and infrared spectra of complex biological material using bovine serum albumin. The performance competes with commercial systems; moreover, it presents the additional advantage of simultaneously operating Raman and infrared techniques. To the best of our knowledge, it is the first demonstration of a combined Raman-infrared system that can analyze the same sample volume and obtain optically sectioned Raman signals. Additionally, quantitative comparison of confocality of backscattering micro-Raman and off-axis Raman was performed for the first time. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
20. Deconvolution microscopy: A platform for rapid on‐site evaluation of fine needle aspiration specimens that enables recovery of the sample.
- Author
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Liao, Haihui, Sheridan, Todd, Cosar, Ediz, Owens, Christopher, Zuo, Tao, Wang, Xiaofei, Akalin, Ali, Kandil, Dina, Dresser, Karen, Fogarty, Kevin, Bellve, Karl, Baer, Christina, and Fischer, Andrew
- Subjects
- *
NEEDLE biopsy , *ON-site evaluation , *MICROSCOPY , *DECONVOLUTION (Mathematics) , *DIAGNOSTIC ultrasonic imaging - Abstract
Context: Rapid on‐site evaluation (ROSE) optimises the performance of cytology, but requires skilled handling, and smearing can make the material unavailable for some ancillary tests. There is a need to facilitate ROSE without sacrificing part of the sample. Objective: We evaluated the image quality of inexpensive deconvolution fluorescence microscopy for optically sectioning non‐smeared fine needle aspiration (FNA) tissue fragments. Design: A portion of residual material from 14 FNA samples was stained for 3 min in Hoechst 33342 and Sypro™ Red to label DNA and protein respectively, transferred to an imaging chamber, and imaged at 200× or 400× magnification at 1 micron intervals using a GE DeltaVision inverted fluorescence microscope. A deconvolution algorithm was applied to remove out‐of‐plane signal, and the resulting images were inverted and pseudocoloured to resemble H&E sections. Five cytopathologists blindly diagnosed 2 to 4 representative image stacks per case (total 70 evaluations), and later compared them to conventional epifluorescent images. Results: Accurate definitive diagnoses were rendered in 45 (64%) of 70 total evaluations; equivocal diagnoses (atypical or suspicious) were made in 21 (30%) of the 70. There were two false positive and two false negative "definite" diagnoses in three cases (4/70; 6%). Cytopathologists preferred deconvolved images compared to raw images (P < 0.01). The imaged fragments were recovered and prepared into a ThinPrep or cell block without discernible alteration. Conclusions: Deconvolution improves image quality of FNA fragments compared to epifluorescence, often allowing definitive diagnosis while enabling the ROSE material to be subsequently triaged. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
21. Optical sectioning of unlabeled samples using brightfield microscopy.
- Author
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Gutiérrez-Medina, Braulio
- Subjects
- *
MICROSCOPY , *DIGITAL image processing , *FLUORESCENCE microscopy , *LIGHT filters , *THREE-dimensional imaging - Abstract
The bright-field (BF) optical microscope is a traditional bioimaging tool that has been recently tested for depth discrimination during evaluation of specimen morphology; however, existing approaches require dedicated instrumentation or extensive computer modeling. We report a direct method for three-dimensional (3D) imaging in BF microscopy, applicable to label-free samples, where we use Köhler illumination in the coherent regime and conventional digital image processing filters to achieve optical sectioning. By visualizing fungal, animal tissue, and plant samples and comparing with light-sheet fluorescence microscopy imaging, we demonstrate the accuracy and applicability of the method, showing how the standard microscope is an effective 3D imaging device. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
22. The adult zebrafish retina: In vivo optical sectioning with Confocal Scanning Laser Ophthalmoscopy and Spectral-Domain Optical Coherence Tomography
- Author
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Bell, Brent A, Yuan, Alex, Dicicco, Rose M, Fogerty, Joseph, Lessieur, Emma M, and Perkins, Brian D
- Subjects
Biomedical and Clinical Sciences ,Ophthalmology and Optometry ,Eye Disease and Disorders of Vision ,Biomedical Imaging ,Bioengineering ,Neurosciences ,Eye ,Animals ,Disease Models ,Animal ,Fluorescein Angiography ,Fundus Oculi ,Ophthalmoscopy ,Reproducibility of Results ,Retinal Diseases ,Retinal Ganglion Cells ,Tomography ,Optical Coherence ,Zebrafish ,Retina ,SLO ,SDOCT ,Optical sectioning ,Morphology ,In vivo ,In vivo ,Medical Biochemistry and Metabolomics ,Opthalmology and Optometry ,Ophthalmology & Optometry ,Ophthalmology and optometry - Abstract
Non-invasive imaging is an invaluable diagnostic tool in ophthalmology. Two imaging devices, the scanning laser ophthalmoscope (SLO) and spectral domain optical coherence tomography (SDOCT), emerged from the clinical realm to provide research scientists with a real-time view of ocular morphology in living animals. We utilized these two independent imaging modalities in a complementary manner to perform in vivo optical sectioning of the adult zebrafish retina. Due to the very high optical power of the zebrafish lens, the confocal depth of field is narrow, allowing for detailed en face views of specific retinal layers, including the cone mosaic. Moreover, we demonstrate that both native reflectance, as well as fluorescent features observed by SLO, can be combined with axial in-depth information obtained by SDOCT. These imaging approaches can be used to screen for ocular phenotypes and monitor retinal pathology in a non-invasive manner.
- Published
- 2016
23. Background Noise Suppression of Optical Sectioning Structured Illumination Microscopy via Fourier Domain Reconstruction
- Author
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Shipei Dang, Jia Qian, Tong Peng, Chen Bai, Junwei Min, Haixia Wang, Baoli Yao, and Dan Dan
- Subjects
structured illumination microscopy (SIM) ,optical sectioning ,background noise suppression ,image reconstruction ,Fourier domain ,Physics ,QC1-999 - Abstract
Optical sectioning structured illumination microscopy (OS-SIM) has been attracting considerable interest in fast 3D microscopy. The reconstruction of optical sectioning images in the conventional method employs the root-mean-square (RMS) algorithm in the spatial domain, which is prone to residual background noise. To overcome this problem, we propose a Fourier domain based optical sectioning image reconstruction algorithm (termed Fourier-OS-SIM), which has an improved background noise suppression capability compared to the RMS algorithm. The experimental results verified the feasibility and the effectiveness of the algorithm. The improved performance of the Fourier-OS-SIM may find more applications in biomedical or industrial fields.
- Published
- 2022
- Full Text
- View/download PDF
24. Epi‐illumination dark‐field microscopy enables direct visualization of unlabeled small organisms with high spatial and temporal resolution.
- Author
-
Shi, Ruijie, Chen, Xiangyang, Huo, Jing, Guo, Siyue, Smith, Zachary J., and Chu, Kaiqin
- Abstract
Dark‐field microscopy is known to offer both high resolution and direct visualization of thin samples. However, its performance and optimization on thick samples is under‐explored and so far, only meso‐scale information from whole organisms has been demonstrated. In this work, we carefully investigate the difference between trans‐ and epi‐illumination configurations. Our findings suggest that the epi‐illumination configuration is superior in both contrast and fidelity compared to trans‐illumination, while having the added advantage of experimental simplicity and an "open top" for experimental intervention. Guided by the theoretical analysis, we constructed an epi‐illumination dark‐field microscope with measured lateral and axial resolutions of 260 nm and 520 nm, respectively. Subcellular structures in whole organisms were directly visualized without the need for image reconstruction, and further confirmed via simultaneous fluorescence imaging. With an imaging speed of 20 to 50 fps, we visualize fast dynamic processes such as cell division and pharyngeal pumping in Caenorhabditis elegans. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
25. Improved spectral imaging microscopy for cultural heritage through oblique illumination
- Author
-
Lindsay Oakley, Stephanie Zaleski, Billie Males, Oliver Cossairt, and Marc Walton
- Subjects
Spectral imaging microscopy ,Oblique illumination ,Optical sectioning ,Surface shape ,Dictionary learning ,Micro analysis ,Fine Arts ,Analytical chemistry ,QD71-142 - Abstract
Abstract This work presents the development of a flexible microscopic chemical imaging platform for cultural heritage that utilizes wavelength-tunable oblique illumination from a point source to obtain per-pixel reflectance spectra in the VIS–NIR range. The microscope light source can be adjusted on two axes allowing for a hemisphere of possible illumination directions. The synthesis of multiple illumination angles allows for the calculation of surface normal vectors, similar to phase gradients, and axial optical sectioning. The extraction of spectral reflectance images with high spatial resolutions from these data is demonstrated through the analysis of a replica cross-section, created from known painting reference materials, as well as a sample extracted from a painting by Pablo Picasso entitled La Miséreuse accroupie (1902). These case studies show the rich microscale molecular information that may be obtained using this microscope and how the instrument overcomes challenges for spectral analysis commonly encountered on works of art with complex matrices composed of both inorganic minerals and organic lakes.
- Published
- 2020
- Full Text
- View/download PDF
26. Optical Sectioning Microscopy Through Single-Shot Lightfield Protocol
- Author
-
E. Sanchez-Ortiga, G. Scrofani, G. Saavedra, and M. Martinez-Corral
- Subjects
Fourier integral microscope ,fourier lightfield microscope ,FiMic ,GPU computing ,lightfield microscope ,optical sectioning ,Electrical engineering. Electronics. Nuclear engineering ,TK1-9971 - Abstract
Optical sectioning microscopy is usually performed by means of a scanning, multi-shot procedure in combination with non-uniform illumination. In this paper, we change the paradigm and report a method that is based in the lightfield concept, and that provides optical sectioning for 3D microscopy images after a single-shot capture. To do this we first capture multiple orthographic perspectives of the sample by means of Fourier-domain integral microscopy (FiMic). The second stage of our protocol is the application of a novel refocusing algorithm that is able to produce optical sectioning in real time, and with no resolution worsening, in the case of sparse fluorescent samples. We provide the theoretical derivation of the algorithm, and demonstrate its utility by applying it to simulations and to experimental data.
- Published
- 2020
- Full Text
- View/download PDF
27. Applying single-molecule localisation microscopy to achieve virtual optical sectioning and study T-cell activation
- Author
-
Palayret, Matthieu Grégoire Simon
- Subjects
540 ,T-cell receptor ,kinetic-segregation ,CD28 super-agonist ,single-molecule localisation microscopy ,super-resolution fluorescence microscopy ,virtual 'light-sheet' ,optical sectioning - Abstract
Single-molecule localisation microscopy (SMLM) allows imaging of fluorescently-tagged proteins in live cells with a precision well below that of the diffraction limit. As a single-molecule technique, it has also introduced a new quantitative approach to fluorescence microscopy. In the Part A of this thesis, the design and building of three SMLM instruments, the implementation of a custom-developed image analysis package and the characterisation of the photo-physical properties of the photo-activable fluorescent protein used in this thesis (mEos), are discussed. Then, a new post-processing method for SMLM analysis is characterised: axial optical sectioning of SMLM images is demonstrated by thresholding fitted localisations using their fitted width and amplitude to reject fluorophores that emit from above or below a virtual ?light-sheet?, a thin volume centred on the focal plane of the microscope. This method provides qualitative and quantitative improvements to SMLM. In the Part B of this thesis, SMLM is applied to study T cell activation. Although the T cell receptor plays a key role in immunity, its stoichiometry in the membrane of resting T cells is still a matter of debate. Here, single-molecule counting methods are implemented to compare the stoichiometry of TCRs fused with mEos2 in resting T cells to monomeric and dimeric controls. However, because of the stochasticity of mEos2 photo-physics, results are inconclusive and new counting techniques based on structural imaging are discussed. In addition to TCR triggering, T cells require the co-stimulatory triggering of the CD28 transmembrane receptor to become fully activated. However, some immobilised anti-CD28 antibodies, referred to as super-agonists (SA), can directly activate T cells without triggering the TCR. In this thesis, single-molecule tracking techniques are used to investigate the molecular mechanism of CD28 super-agonism in live T cells. The results indicate that the diffusion of CD28 is slowed by SA binding. This effect is further discussed in light of the kinetic-segregation model proposed for TCR triggering. Quantitative SMLM as implemented and further developed in this work offers new tools to investigate the molecular mechanisms initiating T cell activation, ultimately facilitating the discovery of novel approaches to target these pathways for therapeutic purposes.
- Published
- 2015
- Full Text
- View/download PDF
28. Improving the depth resolution of STEM-ADF sectioning by 3D deconvolution.
- Author
-
Ishizuka, A, Ishizuka, K, Ishikawa, R, Shibata, N, Ikuhara, Y, Hashiguchi, H, and Sagawa, R
- Subjects
- *
DECONVOLUTION (Mathematics) , *MAXIMUM entropy method , *FAST Fourier transforms , *TRANSMISSION electron microscopes , *SCANNING electron microscopes , *THREE-dimensional imaging - Abstract
Although the possibility of locating single atom in three dimensions using the scanning transmission electron microscope (STEM) has been discussed with the advent of aberration correction technology, it is still a big challenge. In this report we have developed deconvolution routines based on maximum entropy method (MEM) and Richardson–Lucy algorithm (RLA), which are applicable to the STEM-annular dark-field (ADF) though-focus images to improve the depth resolution. The new three-dimensional (3D) deconvolution routines require a limited defocus-range of STEM-ADF images that covers a whole sample and some vacuum regions. Since the STEM-ADF probe is infinitely elongated along the optical axis, a 3D convolution is performed with a two-dimensional (2D) convolution over xy -plane using the 2D fast Fourier transform in reciprocal space, and a one-dimensional convolution along the z-direction in real space. Using our new deconvolution routines, we have processed simulated focal series of STEM-ADF images for single Ce dopants embedded in wurtzite-type AlN. Applying the MEM, the Ce peaks are clearly localized along the depth, and the peak width is reduced down to almost one half. We also applied the new deconvolution routines to experimental focal series of STEM-ADF images of a monolayer graphene. The RLA gives smooth and high-P/B ratio scattering distribution, and the graphene layer can be easily detected. Using our deconvolution algorithms, we can determine the depth locations of the heavy dopants and the graphene layer within the precision of 0.1 and 0.2 nm, respectively. Thus, the deconvolution must be extremely useful for the optical sectioning with 3D STEM-ADF images. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
29. Quantitative Live Cell FLIM Imaging in Three Dimensions
- Author
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Le Marois, Alix, Suhling, Klaus, COHEN, IRUN R., Series editor, LAJTHA, ABEL, Series editor, LAMBRIS, JOHN D., Series editor, PAOLETTI, RODOLFO, Series editor, REZAEI, NIMA, Series editor, and Dmitriev, Ruslan I., editor
- Published
- 2017
- Full Text
- View/download PDF
30. Telecentric design for digital‐scanning‐based HiLo optical sectioning endomicroscopy with an electrically tunable lens.
- Author
-
Hsiao, Haw, Lin, Chen‐Yen, Vyas, Sunil, Huang, Kuang‐Yuh, Yeh, J. Andrew, and Luo, Yuan
- Abstract
Confocal endoscopy has been widely used to obtain fine optically sectioned images. However, confocal endomicroscopic images are formed by point‐by‐point scanning in both lateral and axial directions, which results in long image acquisition time. Here, an endomicroscope with telecentric configuration is presented to achieve nonmechanical and rapid axial scanning for volumetric fluorescence imaging. In our system, optical sectioning in wide‐field fashion is obtained through HiLo imaging with a digital micromirror device. Axial scanning, without mechanical moving parts, is conducted by digital focus adjustment using an electrically tunable lens, offering constant magnification and contrast. We demonstrate imaging performance of our system with optically sectioned images using fluorescently labeled beads, as well as ex vivo mice cardiac tissue samples. Our system provides multiple advantages, in terms of improved scanning range, and reduced image acquisition time, which shows great potentials for three‐dimensional biopsies of volumetric biological samples. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
31. Optical sectioning in the aberration-corrected scanning transmission and scanning confocal electron microscope
- Author
-
Behan, Gavin Joseph and Nellist, Peter
- Subjects
535 ,Materials Sciences ,scanning transmission electron microscopy ,aberration-corrected ,scanning confocal electron microscopy ,optical sectioning - Abstract
This thesis concerns the experimental application of the technique of optical sectioning in the aberration-corrected scanning transmission electron microscope (STEM). Another aim was to perform optical sectioning experiments on the still relatively new scanning confocal electron microscope (SCEM). To test the feasibility of this technique, experiments were performed on a variety of samples to measure the achievable depth response. Deconvolution methods were explored in an attempt to further improve the depth response. Finally, some of the first optical sectioning experiments were performed in the SCEM using both elastic and inelastically scattered electrons. The results showed a clear need to investigate confocal electron microscopy due to the missing cone problem for incoherent imaging in the STEM. This is particularly evident when imaging objects of greater width than the STEM probe. Confocal electron microscopy using inelastic electrons appeared to be a promising imaging mode for the future with this thesis consisting of early work in the field.
- Published
- 2009
32. Comparison of Two- and Three-Beam Interference Pattern Generation in Structured Illumination Microscopy
- Author
-
Jiuling Liao, Lina Liu, Tingai Chen, Xianyuan Xia, Hui Li, and Wei Zheng
- Subjects
structured illumination microscopy ,optical sectioning ,digital micro-mirror device ,multi-beam interference ,Applied optics. Photonics ,TA1501-1820 - Abstract
Structured illumination microscopy (SIM) provides wide-field optical sectioning in the focal plane by modulating the imaging information using fringe pattern illumination. For generating the fringe pattern illumination, a digital micro-mirror device (DMD) is commonly used due to its flexibility and fast refresh rate. However, the benefit of different pattern generation, for example, the two-beam interference mode and the three-beam interference mode, has not been clearly investigated. In this study, we systematically analyze the optical sectioning provided by the two-beam inference mode and the three-beam interference mode of DMD. The theoretical analysis and imaging results show that the two-beam interference mode is suitable for fast imaging of the superficial dynamic target due to reduced number of phase shifts needed to form the image, and the three-beam interference mode is ideal for imaging three-dimensional volume due to its superior optical sectioning by the improved modulation of the illumination patterns. These results, we believe, will provide better guidance for the use of DMD for SIM imaging and also for the choice of beam patterns in SIM application in the future.
- Published
- 2021
- Full Text
- View/download PDF
33. A Two-Photon Microimaging-Microdevice System for Four-Dimensional Imaging of Local Drug Delivery in Tissues
- Author
-
Guigen Liu, Veronica Valvo, Sebastian W. Ahn, Devon Thompson, Kyle Deans, Jeon Woong Kang, Sharath Bhagavatula, Christine Dominas, and Oliver Jonas
- Subjects
biomedical microdevice ,two-photon micro-endoscopy ,optical sectioning ,local drug delivery ,tumors ,in vivo testing ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Advances in the intratumor measurement of drug responses have included a pioneering biomedical microdevice for high throughput drug screening in vivo, which was further advanced by integrating a graded-index lens based two-dimensional fluorescence micro-endoscope to monitor tissue responses in situ across time. While the previous system provided a bulk measurement of both drug delivery and tissue response from a given region of the tumor, it was incapable of visualizing drug distribution and tissue responses in a three-dimensional (3D) way, thus missing the critical relationship between drug concentration and effect. Here we demonstrate a next-generation system that couples multiplexed intratumor drug release with continuous 3D spatial imaging of the tumor microenvironment via the integration of a miniaturized two-photon micro-endoscope. This enables optical sectioning within the live tissue microenvironment to effectively profile the entire tumor region adjacent to the microdevice across time. Using this novel microimaging-microdevice (MI-MD) system, we successfully demonstrated the four-dimensional imaging (3 spatial dimensions plus time) of local drug delivery in tissue phantom and tumors. Future studies include the use of the MI-MD system for monitoring of localized intra-tissue drug release and concurrent measurement of tissue responses in live organisms, with applications to study drug resistance due to nonuniform drug distribution in tumors, or immune cell responses to anti-cancer agents.
- Published
- 2021
- Full Text
- View/download PDF
34. Imaging flowers: a guide to current microscopy and tomography techniques to study flower development.
- Author
-
Prunet, Nathanaël and Duncan, Keith
- Subjects
- *
FLOWER development , *BIOLOGICAL specimens , *MICROSCOPICAL technique , *MICROSCOPY , *PLANT fertilization , *DEVELOPMENTAL biology - Abstract
Developmental biology relies heavily on our ability to generate three-dimensional images of live biological specimens through time, and to map gene expression and hormone response in these specimens as they undergo development. The last two decades have seen an explosion of new bioimaging technologies that have pushed the limits of spatial and temporal resolution and provided biologists with invaluable new tools. However, plant tissues are difficult to image, and no single technology fits all purposes; choosing between many bioimaging techniques is not trivial. Here, we review modern light microscopy and computed projection tomography methods, their capabilities and limitations, and we discuss their current and potential applications to the study of flower development and fertilization. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
35. Improved spectral imaging microscopy for cultural heritage through oblique illumination.
- Author
-
Oakley, Lindsay, Zaleski, Stephanie, Males, Billie, Cossairt, Oliver, and Walton, Marc
- Subjects
- *
SPECTRAL imaging , *MULTISPECTRAL imaging , *CULTURAL property , *SPECTRAL reflectance , *MICROSCOPY , *LIGHT sources , *HIGH resolution imaging - Abstract
This work presents the development of a flexible microscopic chemical imaging platform for cultural heritage that utilizes wavelength-tunable oblique illumination from a point source to obtain per-pixel reflectance spectra in the VIS–NIR range. The microscope light source can be adjusted on two axes allowing for a hemisphere of possible illumination directions. The synthesis of multiple illumination angles allows for the calculation of surface normal vectors, similar to phase gradients, and axial optical sectioning. The extraction of spectral reflectance images with high spatial resolutions from these data is demonstrated through the analysis of a replica cross-section, created from known painting reference materials, as well as a sample extracted from a painting by Pablo Picasso entitled La Miséreuse accroupie (1902). These case studies show the rich microscale molecular information that may be obtained using this microscope and how the instrument overcomes challenges for spectral analysis commonly encountered on works of art with complex matrices composed of both inorganic minerals and organic lakes. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
36. Multiplane HiLo microscopy with speckle illumination and non-local means denoising.
- Author
-
Zheng, Shuqi, Koyama, Minoru, and Mertz, Jerome
- Subjects
- *
MICROSCOPY , *IMAGE denoising , *NOISE control , *LIGHTING , *CARDIAC imaging , *SPECKLE interferometry - Abstract
HiLo microscopy synthesizes an optically sectioned image from two images, one obtained with uniform and another with patterned illumination, such as laser speckle. Speckle-based HiLo has the advantage of being robust to aberrations but is susceptible to residual speckle noise that is difficult to control. We present a computational method to reduce this residual noise without undermining resolution. In addition, we improve the versatility of HiLo microscopy by enabling simultaneous multiplane imaging (here nine planes). Our goal is to perform fast, high-contrast, multiplane imaging with a conventional camera-based fluorescence microscope. Multiplane HiLo imaging is achieved with the use of a single camera and z-splitter prism. Speckle noise reduction is based on the application of a non-local means (NLM) denoising method to perform ensemble averaging of speckle grains. We demonstrate the capabilities of multiplane HiLo with NLM denoising both with synthesized data and by imaging cardiac and brain activity in zebrafish larvae at 40 Hz frame rates. Multiplane HiLo microscopy aided by NLM denoising provides a simple tool for fast optically sectioned volumetric imaging that can be of general utility for fluorescence imaging applications. [ABSTRACT FROM AUTHOR]
- Published
- 2023
- Full Text
- View/download PDF
37. Digital Incoherent Compressive Holography Using a Geometric Phase Metalens
- Author
-
Jonghyun Lee, Youngrok Kim, Kihong Choi, Joonku Hahn, Sung-Wook Min, and Hwi Kim
- Subjects
diffractive optics ,digital holography ,computer-generated hologram ,metasurface ,compressive sensing ,optical sectioning ,Chemical technology ,TP1-1185 - Abstract
We propose a compressive self-interference incoherent digital holography (SIDH) with a geometric phase metalens for section-wise holographic object reconstruction. We specify the details of the SIDH with a geometric phase metalens design that covers the visible wavelength band, analyze a spatial distortion problem in the SIDH and address a process of a compressive holographic section-wise reconstruction with analytic spatial calibration. The metalens allows us to realize a compressive SIDH system in the visible wavelength band using an image sensor with relatively low bandwidth. The operation of the proposed compressive SIDH is verified through numerical simulations.
- Published
- 2021
- Full Text
- View/download PDF
38. Confocal Laser-scanning Microscopy in Filamentous Fungi
- Author
-
Mouriño-Pérez, Rosa R., Roberson, Robert W., Gupta, Vijai Kumar, Series editor, Tuohy, Maria G., Series editor, Dahms, Tanya E. S., editor, and Czymmek, Kirk J., editor
- Published
- 2015
- Full Text
- View/download PDF
39. Optimization of structured illumination microscopy with designing and rotating a grid pattern using a spatial light modulator.
- Author
-
Jeong-Heon Han, Nak-Won Yoo, Ji-Hoon Kang, Byeong-Kwon Ju, and Min-Chul Park
- Subjects
- *
SPATIAL light modulators , *NUMERICAL apertures , *MICROSCOPY , *FREQUENCY spectra , *LIGHTING - Abstract
Our structured illumination microscopy (SIM) is based on a spatial light modulator (SLM) instead of an illumination mask, which does not need to be attached to a linear stage. This SIM can easily design the period of the one-dimensional grid related to the optical sectioning strength and can rapidly acquire three-dimensional data. The optimization of SIM with an SLM is proposed. Previous studies primarily varied magnification with a high numerical aperture objective to optimize the axial response. It is feasible to obtain the maximum optical sectioning strength by designing a grid pattern that has an appropriately high spatial frequency and to uniformly cover the entire frequency spectrum of the sample by rotating a grid pattern. We have successfully optimized SIM with such a grid and covered the frequency spectrum by rotating a grid pattern in multiple orientations. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
40. Non-Destructive Reflectance Mapping of Collagen Fiber Alignment in Heart Valve Leaflets.
- Author
-
Goth, Will, Potter, Sam, Allen, Alicia C. B., Zoldan, Janet, Sacks, Michael S., and Tunnell, James W.
- Abstract
Collagen fibers are the primary structural elements that define many soft-tissue structure and mechanical function relationships, so that quantification of collagen organization is essential to many disciplines. Current tissue-level collagen fiber imaging techniques remain limited in their ability to quantify fiber organization at macroscopic spatial scales and multiple time points, especially in a non-contacting manner, requiring no modifications to the tissue, and in near real-time. Our group has previously developed polarized spatial frequency domain imaging (pSFDI), a reflectance imaging technique that rapidly and non-destructively quantifies planar collagen fiber orientation in superficial layers of soft tissues over large fields-of-view. In this current work, we extend the light scattering models and image processing techniques to extract a critical measure of the degree of collagen fiber alignment, the normalized orientation index (NOI), directly from pSFDI data. Electrospun fiber samples with architectures similar to many collagenous soft tissues and known NOI were used for validation. An inverse model was then used to extract NOI from pSFDI measurements of aortic heart valve leaflets and clearly demonstrated changes in degree of fiber alignment between opposing sides of the sample. These results show that our model was capable of extracting absolute measures of degree of fiber alignment in superficial layers of heart valve leaflets with only general a priori knowledge of fiber properties, providing a novel approach to rapid, non-destructive study of microstructure in heart valve leaflets using a reflectance geometry. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
41. Spatially-incoherent annular illumination microscopy for bright-field optical sectioning.
- Author
-
Ma, Xiao, Zhang, Zibang, Yao, Manhong, Peng, Junzheng, and Zhong, Jingang
- Subjects
- *
MICROSCOPY , *WAVELENGTHS , *LIGHT emitting diodes , *LIGHTING , *NUMERICAL apertures - Abstract
Highlights • An annular LED array for oblique illumination with high brightness, uniform lighting is first proposed. • The proposed technique can achieve non-fluorescence and bright-field optical sectioning. • The proposed technique is able to simultaneously improve lateral and axial resolution. Using a 525-nm wavelength LED array the proposed technique can provide 600-nm axial resolution and 150-nm lateral resolution. • The proposed technique is of simplicity, low cost, but high performance and good compatibility. It can be implemented by simply adapting a standard bright-field microscope. • Different from almost all advanced microscopies for optical sectioning, the proposed microscopy is based on direct imaging (non-computational imaging). It allows one to acquire sectioning images through a camera or an eyepiece in real time. Abstract Numerous advanced microscopic imaging techniques have been proposed for optical sectioning, but they generally employ a complex and costly optical system. Here we report a microscopy termed spatially-incoherent annular illumination microscopy (SAIM). It allows for simple, effective, non-fluorescence, and bright-field optical sectioning. The proposed technique is implemented by installing an annular array of light emitting diodes (LEDs) on a standard bright-field microscope for illumination. The LED array produces distinctive illumination, that is, each LED provides coherent, large-angle oblique illumination while all LEDs generate spatially-incoherent annular illumination. Such a distinctive illumination can improve both lateral resolution and axial resolution. The improvement of lateral resolution is due to the coherent and large-angle oblique illumination. The spatially-incoherent annular illumination can improve the axial resolution. It is because, for defocused structures, each LED results in a blurred image with a different lateral shift and all LEDs result in an incoherent stagger superposition of the defocused images. The superposition looks much more blurred, which improves the contrast of the in-focus image remarkably. We experimentally demonstrate that SAIM is able to provide bright-field optical sections with 600-nm axial resolution and 150-nm lateral half-pitch resolution by using a 525-nm wavelength LED array and an objective with 100X, numerical aperture (NA) 1.25. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
42. Optical sectioning in multifoci Raman hyperspectral imaging.
- Author
-
Liao, Zhiyu, Sinjab, Faris, Elsheikha, Hany M., and Notingher, Ioan
- Subjects
- *
HYPERSPECTRAL imaging systems , *RAMAN spectroscopy , *LIGHT modulators , *LIQUID crystals , *LASER beams - Abstract
In this study, we compared the depth discrimination and speed performance of multifoci Raman hyperspectral imaging with the reference standard of a single laser point confocal Raman mapping. A liquid crystal spatial light modulator was employed for the generation of multifoci laser beams, and a digital micromirror device was used as a software‐configurable reflective pinhole array. The patterns of the laser foci and pinhole array can be rapidly changed without requiring any hardware alterations. Confocal patterns with different distance‐to‐size ratios were tested and compared. After optimization of the laser‐foci pattern, we demonstrated the feasibility of multifoci Raman hyperspectral microscopy for recording depth‐resolved molecular maps of biological cells (Acanthamoeba castellanii trophozoites). Micrometric depth discrimination and short acquisition times (20 min for single plane confocal image) were achieved. A liquid crystal spatial light modulator was employed for the generation of multifoci laser beams, and a digital micromirror device was used as a software‐configurable reflective pinhole array. Confocal patterns with different distance‐to‐size ratios were tested to achieve depth‐resolved Raman molecular maps of biological cells. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
43. Parallel multi-slit modulation and decoding technique for high-resolution surface measurement in structured illumination microscopy.
- Author
-
Chai, Changchun, Chen, Cheng, Huang, Jinkang, You, Wu, Wang, Shuai, Yang, Wenjun, Liu, Xiaojun, and Lei, Zili
- Subjects
- *
SURFACE topography measurement , *DECODING algorithms , *LIGHTING , *MICROSCOPY , *SURFACE topography - Abstract
• Novel parallel multi-slit modulation and decoding technique for brightfield reflection optically-sectioned structured illumination microscopy. • Specially-designed decoding algorithm based on out-of-focus background estimation ensures high-quality optical sectioning independent of illumination pattern frequency. • Incoherent structured illumination microscopy provides consistent high-resolution measurements for a wide range of engineering surface topographies. Optically-sectioned structured illumination microscopy (OS-SIM) is an important tool for biological imaging and engineering surface measurements. However, in the current OS-SIM systems, the dependence of the sectioning strength on illumination pattern frequency hinders the achievement of consistent high axial resolution for various surface topography measurements. In this paper, we develop a parallel multi-slit modulation and decoding technique for OS-SIM, called PMMD-OS-SIM, to solve the existing dependence problem. Specifically, a set of high-contrast parallel multi-slit illumination patterns are projected onto the sample to modulate the surface height information. And then, a specially-designed decoding algorithm is applied to the modulated patterns for high-quality optical sectioning. By effectively combining the above modulation and decoding techniques, the optical-sectioning strength of PMMD-OS-SIM is decoupled from the illumination pattern frequency, thereby facilitating consistent high-resolution measurements for a wide range of surface topographies. The validity of the proposed method is demonstrated by measurement experiments performed on various test samples. [ABSTRACT FROM AUTHOR]
- Published
- 2023
- Full Text
- View/download PDF
44. Advances in nonlinear optical microscopy techniques for in vivo and in vitro neuroimaging
- Author
-
Subir Das, Joel Markus Vaz, Sparsha Pallen, Nirmal Mazumder, and Yuthika Shetty
- Subjects
Materials science ,Optical sectioning ,Harmonic generation ,Mechanism (biology) ,Coherent Raman scattering ,Biophysics ,Neuroimaging ,Review ,Optical microscopy ,Nonlinear optical microscopy ,Multiphoton fluorescence microscopy ,Optical imaging ,Structural Biology ,In vivo ,Molecular Biology ,Neuroscience - Abstract
Understanding the mechanism of the brain via optical microscopy is one of the challenges in neuroimaging, considering the complex structures. Advanced neuroimaging techniques provide a more comprehensive insight into patho-mechanisms of brain disorders, which is useful in the early diagnosis of the pathological and physiological changes associated with various neurodegenerative diseases. Recent advances in optical microscopy techniques have evolved powerful tools to overcome scattering of light and provide improved in vivo neuroimaging with sub-cellular resolution, endogenous contrast specificity, pinhole less optical sectioning capability, high penetration depth, and so on. The following article reviews the developments in various optical imaging techniques including two-photon and three-photon fluorescence, second-harmonic generation, third-harmonic generation, coherent anti-Stokes Raman scattering, and stimulated Raman scattering in neuroimaging. We have outlined the potentials and drawbacks of these techniques and their possible applications in the investigation of neurodegenerative diseases.
- Published
- 2021
45. 3D Structured Illumination Microscopy via Channel Attention Generative Adversarial Network
- Author
-
Siwei Zhang, Yuting Guo, Di Li, Dong Li, Qionghai Dai, Xingye Chen, and Chang Qiao
- Subjects
Channel (digital image) ,Optical sectioning ,Computer science ,business.industry ,Structured illumination microscopy ,02 engineering and technology ,Iterative reconstruction ,Photobleaching ,Atomic and Molecular Physics, and Optics ,020210 optoelectronics & photonics ,Live cell imaging ,Microscopy ,0202 electrical engineering, electronic engineering, information engineering ,Computer vision ,Artificial intelligence ,Electrical and Electronic Engineering ,business ,Image resolution - Abstract
Three-dimensional (3D) structured illumination microscopy (SIM) plays an important role in biological volumetric imaging with the capabilities of doubling the lateral and axial resolution and optical sectioning. However, 3D-SIM suffers from more photobleaching and phototoxicity compared to other volumetric imaging modalities, such as light-sheet microscopy, because it requires 15 raw images per axial slice, which hampers its widespread application in live cell imaging. Here we report the design of a channel attention generative adversarial network (caGAN) that improves the quality of 3D-SIM reconstruction under low signal-to-noise-ratio (SNR) condition and enables reconstruction using fewer raw images. Compared to the conventional algorithm, caGAN-SIM achieves comparable or higher reconstruction fidelity while using 15-fold less signal level. We demonstrate the superior performance of caGAN-SIM for various subcellular structures and its ability in long-term multi-color 3D super-resolution imaging using the example of dynamic interactions between microtubules and lysosomes in live cells.
- Published
- 2021
46. Nonscanning three-dimensional measurement by structured illumination sectioning microscopy.
- Author
-
Tong Wang, Tao Liu, Shuming Yang, Qiang Liu, and Yiming Wang
- Subjects
- *
MICROSCOPY , *LIGHTING , *MICROELECTRONICS - Abstract
To achieve nonscanning three-dimensional measurement of microstructures, the optically sectioning mechanism has been used based on the structured illumination microscopy. The key point is to implement nonscanning measurement within a limited axial range according to the measured one-sided linear response curve. One-dimensional transmission grating pattern was projected onto the sample, and optically sectioned images were extracted from three-step phase-shifting manipulation. Compared with the basic and differential confocal microscopy, the described method does not need time-consuming axial scanning, so it greatly improves the measurement efficiency. The results show that the described method is useful in the fields of micromechanics, microelectronics, and biomedicine. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
47. Super-resolution structured illumination in optically thick specimens without fluorescent tagging.
- Author
-
Hoffman, Zachary R. and DiMarzio, Charles A.
- Subjects
- *
LIGHT sources , *MICROSCOPY , *SKIN cancer diagnosis , *NUMERICAL apertures , *LIGHT emitting diodes - Abstract
This research extends the work of Hoffman et al. to provide both sectioning and super-resolution using random patterns within thick specimens. Two methods of processing structured illumination in reflectance have been developed without the need for a priori knowledge of either the optical system or the modulation patterns. We explore the use of two deconvolution algorithms that assume either Gaussian or sparse priors. This paper will show that while both methods accomplish their intended objective, the sparse priors method provides superior resolution and contrast against all tested targets, providing anywhere from ∼1.6× to ∼2× resolution enhancement. The methods developed here can reasonably be implemented to work without a priori knowledge about the patterns or point spread function. Further, all experiments are run using an incoherent light source, unknown random modulation patterns, and without the use of fluorescent tagging. These additional modifications are challenging, but the generalization of these methods makes them prime candidates for clinical application, providing super-resolved noninvasive sectioning in vivo. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
48. Line-scanning confocal microendoscope for nuclear morphometry imaging.
- Author
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Yubo Tang, Carns, Jennifer, and Richards-Kortum, Rebecca R.
- Subjects
- *
CONFOCAL fluorescence microscopy , *MOLECULAR probes , *ALGORITHMS , *DIAGNOSIS , *CELL morphology - Abstract
Fiber-optic endomicroscopy is a minimally invasive method to image cellular morphology in vivo. Using a coherent fiber bundle as an image relay, it allows additional imaging optics to be placed at the distal end of the fiber outside the body. In this research, we use this approach to demonstrate a compact, low-cost linescanning confocal fluorescence microendoscope that can be constructed for <$5000. Confocal imaging is enabled without the need for mechanical scanning by synchronizing a digital light projector with the rolling shutter of a CMOS camera. Its axial performance is characterized in comparison with a nonscanned high-resolution microendoscope. We validate the optical sectioning capability of the microendoscope by imaging a two-dimensional phantom and ex vivo mouse esophageal and colon tissues. Results show that optical sectioning using this approach improves visualization of nuclear morphometry and suggest that this low-cost line-scanning microendoscope can be used to evaluate various pathological conditions. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
49. 3D elemental mapping with nanometer scale depth resolution via electron optical sectioning
- Author
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Nellist, Peter [Daresbury Lab., Warrington (United Kingdom). EPSRC SuperSTEM Facility; Univ. of Oxford (United Kingdom). Dept. of Materials]
- Published
- 2016
- Full Text
- View/download PDF
50. Multi-photon excitation imaging of dynamic processes in living cells and tissues
- Author
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Benninger, R. K. P., Hao, M., and Piston, D. W.
- Published
- 2008
- Full Text
- View/download PDF
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