The DNA damage response (DDR) is a fundamental readout for evaluating efficacy of cancer therapeutics, many of which target DNA associated processes. Current techniques to evaluate DDR rely on ...immunostaining for phosphorylated histone H2AX (γ-H2AX), which is an indicator of DNA double-strand breaks. While γ-H2AX immunostaining can provide a snapshot of DDR in fixed cell and tissue samples, this method is technically cumbersome due to temporal monitoring of DDR requiring timepoint replicates, extensive assay development efforts for 3D cell culture samples such as organoids, and time-consuming protocols for γ-H2AX immunostaining and its evaluation. The goal of this current study is to reduce overall burden on assay duration and development in non-small cell lung cancer (NSCLC) organoids by leveraging label-free multiphoton imaging. In this study, simultaneous label-free autofluorescence multiharmonic (SLAM) microscopy was used to provide rich intracellular information based on endogenous contrasts. SLAM microscopy enables imaging of live samples eliminating the need to generate sacrificial sample replicates and has improved image acquisition in 3D space over conventional confocal microscopy. Predictive modeling between label-free SLAM microscopy and γ-H2AX immunostained images confirmed strong correlation between SLAM image features and γ-H2AX signal. Across multiple DNA targeting chemotherapeutics and multiple patient-derived NSCLC organoid lines, the optical redox ratio and third harmonic generation channels were used to robustly predict DDR. Imaging via SLAM microscopy can be used to more rapidly predict DDR in live 3D NSCLC organoids with minimal sample handling and without labeling.
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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.
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.
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.
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.
The photothermal (PT) signal arises from slight changes of the index of refraction in a sample due to absorption of a heating light beam. Refractive index changes are measured with a second probing ...beam, usually of a different color. In the past two decades, this all-optical detection method has reached the sensitivity of single particles and single molecules, which gave birth to original applications in material science and biology. PT microscopy enables shot-noise-limited detection of individual nanoabsorbers among strong scatterers and circumvents many of the limitations of fluorescence-based detection. This review describes the theoretical basis of PT microscopy, the methodological developments that improved its sensitivity toward single-nanoparticle and single-molecule imaging, and a vast number of applications to single-nanoparticle imaging and tracking in material science and in cellular biology.
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.
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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