During cancer resection surgeries, intraoperative histopathologic examination of the surgical specimen is crucial for tumor margin identification. A conventional frozen‐section analysis requires ...complex tissue processing, which prolongs surgery and potentially introduces interpretation errors. Here, as a novel approach to label‐free intraoperative histopathology, a high‐speed reflection‐mode ultraviolet photoacoustic microscopy (UV‐PAM) system employing a waterproof 1‐axis microelectromechanical systems scanner is demonstrated. Label‐free nuclear imaging is photoacoustically verified using tissue sections excised from mice and humans. Moreover, by imaging clinical specimens from cancer patients and numerically quantifying the histopathologic results, it is successfully demonstrated that the proposed UV‐PAM system has great potential as an alternative intraoperative histopathology method with minimal tissue preparation processes.
Here, high‐speed, label‐free, and non‐destructive intraoperative histopathology is introduced with the first PA clinical trials according to the diagnostic grades of thick cancer specimens, demonstrating the multi‐feature analysis using a supervised learning algorithm. This approach has the potential for pathologists to differentiate suspicious cancer region accurately, which can be linked to subsequent postoperative examination.
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2.
Roadmap on Label‐Free Super‐Resolution Imaging Astratov, Vasily N.; Sahel, Yair Ben; Eldar, Yonina C. ...
Laser & photonics reviews,
December 2023, Volume:
17, Issue:
12
Journal Article
Peer reviewed
Open access
Label‐free super‐resolution (LFSR) imaging relies on light‐scattering processes in nanoscale objects without a need for fluorescent (FL) staining required in super‐resolved FL microscopy. The ...objectives of this Roadmap are to present a comprehensive vision of the developments, the state‐of‐the‐art in this field, and to discuss the resolution boundaries and hurdles that need to be overcome to break the classical diffraction limit of the label‐free imaging. The scope of this Roadmap spans from the advanced interference detection techniques, where the diffraction‐limited lateral resolution is combined with unsurpassed axial and temporal resolution, to techniques with true lateral super‐resolution capability that are based on understanding resolution as an information science problem, on using novel structured illumination, near‐field scanning, and nonlinear optics approaches, and on designing superlenses based on nanoplasmonics, metamaterials, transformation optics, and microsphere‐assisted approaches. To this end, this Roadmap brings under the same umbrella researchers from the physics and biomedical optics communities in which such studies have often been developing separately. The ultimate intent of this paper is to create a vision for the current and future developments of LFSR imaging based on its physical mechanisms and to create a great opening for the series of articles in this field.
This Roadmap presents a comprehensive vision of developments in the field of nanoscale imaging of non‐fluorescent objects with a focus on methods allowing to overcome the classical diffraction limit. The scope of this Roadmap spans from diffraction‐limited interference detection techniques to super‐resolution methods based on information science, structured illumination, near‐field, nonlinear, and transformation optics, and advanced superlens designs.
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Cell biologists have long sought the ability to observe intracellular structures in living cells without labels. This study presents procedures to adjust a commercially available apodized ...phase-contrast (APC) microscopy system for better visualizing the dynamic behaviors of various subcellular organelles in living cells. By harnessing the versatility of this technique to capture sequential images, we could observe morphological changes in cellular geometry after virus infection in real time without probes or invasive staining. The tune-up APC microscopy system is a highly efficient platform for simultaneously observing the dynamic behaviors of diverse subcellular structures with exceptional resolution.Key words: Label-free imaging, Organelle dynamics, Virus infections, Apodized phase contrast
Imaging and quantification of nanoparticles in single cells in their most natural condition are expected to facilitate the biotechnological applications of nanoparticles and allow for better ...assessment of their biosafety risks. However, current imaging modalities either require tedious sample preparation or only apply to nanoparticles with specific physicochemical characteristics. Here, the emerging hyperspectral stimulated Raman scattering (SRS) microscopy, as a label‐free and nondestructive imaging method, is used for the first time to investigate the subcellular distribution of nanoparticles in the protozoan Tetrahymena thermophila. The two frequently studied nanoparticles, polyacrylate‐coated α‐Fe2O3 and TiO2, are found to have different subcellular distribution pattern as a result of their dissimilar uptake routes. Significant uptake competition between these two types of nanoparticles is further discovered, which should be paid attention to in future bioapplications of nanoparticles. Overall, this study illustrates the great promise of hyperspectral SRS as an analytical imaging tool in nanobiotechnology and nanotoxicology.
As a label‐free imaging technique, hyper‐spectral stimulated Raman scattering allows the determination of the distribution of dissimilar nanoparticles in single cells or unicellular organisms. Significant uptake competition between dissimilar nanoparticles is also revealed by this method, which should be considered not only in medical/biological applications but also in safety assessments of nanoparticles.
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Imaging of nucleic acids is important for studying cellular processes such as cell division and apoptosis. A noninvasive label‐free technique is attractive. Raman spectroscopy provides rich chemical ...information based on specific vibrational peaks. However, the signal from spontaneous Raman scattering is weak and long integration times are required, which drastically limits the imaging speed when used for microscopy. Coherent Raman scattering techniques, comprising coherent anti‐Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopy, overcome this problem by enhancing the signal level by up to five orders of magnitude. CARS microscopy suffers from a nonresonant background signal, which distorts Raman spectra and limits sensitivity. This makes CARS imaging of weak transitions in spectrally congested regions challenging. This is especially the case in the fingerprint region, where nucleic acids show characteristic peaks. The recently developed SRS microscopy is free from these limitations; excitation spectra are identical to those of spontaneous Raman and sensitivity is close to shot‐noise limited. Herein we demonstrate the use of SRS imaging in the fingerprint region to map the distribution of nucleic acids in addition to proteins and lipids in single salivary gland cells of Drosophila larvae, and in single mammalian cells. This allows the imaging of DNA condensation associated with cell division and opens up possibilities of imaging such processes in vivo.
Imaging nucleic acids: Label‐free optical imaging of nucleic acids in live cells is demonstrated using highly sensitive stimulated Raman scattering (SRS) microscopy. This technique allows in vivo imaging of DNA condensation associated with cell division.
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Whispering‐gallery‐mode microresonators enable materials for single‐molecule label‐free detection and imaging because of their high sensitivity to their microenvironment. However, fabrication and ...materials challenges prevent scalability and limit functionality. All‐glass on‐chip microresonators significantly reduce these difficulties. Construction of all‐glass toroidal microresonators with high quality factor and low mode volume is reported and these are used as platforms for label‐free single‐particle imaging.
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•Myelin fluorescence imaging aids in diagnosing myelin-related diseases and understanding myelin biological processes.•Five kinds of fluorescence imaging techniques and their design ...strategy were summarized and discussed.•The challenges and future development of fluorescence imaging technology in improving myelin visualization are forwarded.
Myelin is an important component of the central nervous system, formed by glial cells surrounding neurons. Its damage is closely associated with diseases, such as multiple sclerosis and white matter malnutrition, making the myelin a potentialized target. Therefore, conducting myelin imaging highly benefits for the early diagnosis and treatment of related diseases. Fluorescence imaging has advantages such as high sensitivity, high specificity, and high signal-to-noise ratio, which can effectively image the myelin and indicate changes in its content. This review summarizes fluorescence imaging techniques used for myelin imaging in recent decades, focusing on the principles and applications of these techniques, and purposing prospects for future development. We hope that the development of fluorescence imaging techniques will provide researchers with new insights into the structure and distribution of myelin, as well as powerful research tools for studying key processes such as nervous system development, degeneration, and regeneration.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The Compact Morpho‐Molecular Microscopy (CM3) is proposed and demonstrated for multi‐functional imaging and characterization of living cells and material structures by simultaneously offering ...quantitative phase imaging (QPI), dispersion characterization, and fluorescence imaging. The compactness and stability of CM3 are realized by propagating lasers of different wavelengths in specially treated optical fibers and fiber‐based beam splitters and wavelength division multiplexers, as well as simplifying the detection scheme through Fourier‐space multiplexing and implementing a single camera for both QPI and fluorescence imaging. Quantitative phase maps of two wavelengths are retrieved from a multiplexed interferogram, and a synthesized wavelength phase map is derived to guide the height profiling of samples with extended depth. With the wavelength resolved phase maps, a physical model is derived to obtain sample dispersion parameters, which further enables us to quantify the hemoglobin concentration of red blood cells in real time. By inserting an appropriate emission color filter, fluorescence imaging is realized using the same camera, which significantly broadens the cell imaging applications of CM3. As a cost‐effective and multifaceted imaging method, CM3 may find many promising applications in live‐cell imaging and material characterization.
For a comprehensive investigation of biological and material structures, Compact Morpho‐Molecular Microscopy is proposed. The article shows the characterization of living cells and materials by simultaneously offering quantitative phase imaging, dispersion characterization, and fluorescence imaging. The method can also achieve high‐speed mapping of sample properties and imaging samples with large thicknesses.
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9.
Advances in Imaging Plant Cell Walls Zhao, Yuanyuan; Man, Yi; Wen, Jialong ...
Trends in plant science,
September 2019, 2019-09-00, 20190901, Volume:
24, Issue:
9
Journal Article
Peer reviewed
Understanding of cell wall architecture, including the crosslinking of cell wall polymers, provides crucial information for elucidating the relationship between cell wall structure and cell function. ...Moreover, examination of the cell wall informs efforts to improve biomass breakdown in bioreactor conditions. Over the past decades, imaging techniques have been used extensively to reveal the structural organization and chemical composition of cell walls, but detailed imaging of the native composition and architecture of the cell wall remains challenging. Here, we review progress in the development of cell wall imaging techniques. In particular, we focus on several advanced, label-free techniques for imaging cell walls and their potential applications in investigation of the biological functions of plant cell walls.
Cell wall imaging can considerably permit direct visualization of the molecular architecture of cell walls and provide detailed chemical information on wall polymers, which is becoming one of the hot topics in contemporary botanical research.Label-free techniques based on Raman spectroscopic imaging, SRS in particular, can provide label-free dynamics and quantitative microanalysis of chemical compositions of living plant cell walls with a characteristic molecular vibration in situ, which has opened exciting new avenues for cell wall imagingFurther studies integrating advanced label-free imaging techniques, using super-resolution microscopy, along with real-time studies of structural changes, will be conductive to refine current molecular organization and better understanding of cell wall architecture.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
In the field of tissue engineering, 3D scaffolds and cells are often combined to yield constructs that are used as therapeutics to repair or restore tissue function in patients. Viable cells are ...often required to achieve the intended mechanism of action for the therapy, where the live cells may build new tissue or may release factors that induce tissue regeneration. Thus, there is a need to reliably measure cell viability in 3D scaffolds as a quality attribute of a tissue‐engineered medical product. Here, we developed a noninvasive, label‐free, 3D optical coherence tomography (OCT) method to rapidly (2.5 min) image large sample volumes (1 mm3) to assess cell viability and distribution within scaffolds. OCT imaging was assessed using a model scaffold‐cell system consisting of a polysaccharide‐based hydrogel seeded with human Jurkat cells. Four test systems were used: hydrogel seeded with live cells, hydrogel seeded with heat‐shocked or fixed dead cells and hydrogel without any cells. Time series OCT images demonstrated changes in the time‐dependent speckle patterns due to refractive index (RI) variations within live cells that were not observed for pure hydrogel samples or hydrogels with dead cells. The changes in speckle patterns were used to generate live‐cell contrast by image subtraction. In this way, objects with large changes in RI were binned as live cells. Using this approach, on average, OCT imaging measurements counted 326 ± 52 live cells per 0.288 mm3 for hydrogels that were seeded with 288 live cells (as determined by the acridine orange‐propidium iodide cell counting method prior to seeding cells in gels). Considering the substantial uncertainties in fabricating the scaffold‐cell constructs, such as the error from pipetting and counting cells, a 13% difference in the live‐cell count is reasonable. Additionally, the 3D distribution of live cells was mapped within a hydrogel scaffold to assess the uniformity of their distribution across the volume. Our results demonstrate a real‐time, noninvasive method to rapidly assess the spatial distribution of live cells within a 3D scaffold that could be useful for assessing tissue‐engineered medical products.
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