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Nature Methods Mar 2015Specific labeling of biomolecules with bright fluorophores is the keystone of fluorescence microscopy. Genetically encoded self-labeling tag proteins can be coupled to...
Specific labeling of biomolecules with bright fluorophores is the keystone of fluorescence microscopy. Genetically encoded self-labeling tag proteins can be coupled to synthetic dyes inside living cells, resulting in brighter reporters than fluorescent proteins. Intracellular labeling using these techniques requires cell-permeable fluorescent ligands, however, limiting utility to a small number of classic fluorophores. Here we describe a simple structural modification that improves the brightness and photostability of dyes while preserving spectral properties and cell permeability. Inspired by molecular modeling, we replaced the N,N-dimethylamino substituents in tetramethylrhodamine with four-membered azetidine rings. This addition of two carbon atoms doubles the quantum efficiency and improves the photon yield of the dye in applications ranging from in vitro single-molecule measurements to super-resolution imaging. The novel substitution is generalizable, yielding a palette of chemical dyes with improved quantum efficiencies that spans the UV and visible range.
Topics: Azetidines; Chemistry Techniques, Synthetic; Coumarins; Fluorescein; Fluorescent Dyes; HeLa Cells; Humans; Microscopy, Ultraviolet; Models, Molecular; Molecular Imaging; Quantum Theory; Rhodamines; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Structure-Activity Relationship
PubMed: 25599551
DOI: 10.1038/nmeth.3256 -
Proceedings of the National Academy of... Jun 2020Hematological analysis, via a complete blood count (CBC) and microscopy, is critical for screening, diagnosing, and monitoring blood conditions and diseases but requires...
Hematological analysis, via a complete blood count (CBC) and microscopy, is critical for screening, diagnosing, and monitoring blood conditions and diseases but requires complex equipment, multiple chemical reagents, laborious system calibration and procedures, and highly trained personnel for operation. Here we introduce a hematological assay based on label-free molecular imaging with deep-ultraviolet microscopy that can provide fast quantitative information of key hematological parameters to facilitate and improve hematological analysis. We demonstrate that this label-free approach yields 1) a quantitative five-part white blood cell differential, 2) quantitative red blood cell and hemoglobin characterization, 3) clear identification of platelets, and 4) detailed subcellular morphology. Analysis of tens of thousands of live cells is achieved in minutes without any sample preparation. Finally, we introduce a pseudocolorization scheme that accurately recapitulates the appearance of cells under conventional staining protocols for microscopic analysis of blood smears and bone marrow aspirates. Diagnostic efficacy is evaluated by a panel of hematologists performing a blind analysis of blood smears from healthy donors and thrombocytopenic and sickle cell disease patients. This work has significant implications toward simplifying and improving CBC and blood smear analysis, which is currently performed manually via bright-field microscopy, and toward the development of a low-cost, easy-to-use, and fast hematological analyzer as a point-of-care device and for low-resource settings.
Topics: Blood Cell Count; Blood Cells; Equipment Design; Humans; Microscopy, Ultraviolet; Molecular Imaging; Point-of-Care Systems
PubMed: 32561645
DOI: 10.1073/pnas.2001404117 -
Scientific Reports Jul 2018Ultraviolet (UV) spectroscopy is a powerful tool for quantitative (bio)chemical analysis, but its application to molecular imaging and microscopy has been limited. Here...
Ultraviolet (UV) spectroscopy is a powerful tool for quantitative (bio)chemical analysis, but its application to molecular imaging and microscopy has been limited. Here we introduce ultraviolet hyperspectral interferometric (UHI) microscopy, which leverages coherent detection of optical fields to overcome significant challenges associated with UV spectroscopy when applied to molecular imaging. We demonstrate that this method enables quantitative spectral analysis of important endogenous biomolecules with subcellular spatial resolution and sensitivity to nanometer-scaled structures for label-free molecular imaging of live cells.
PubMed: 29967322
DOI: 10.1038/s41598-018-28208-0 -
Biomedical Optics Express Oct 2021Neutropenia is a condition identified by an abnormally low number of neutrophils in the bloodstream and signifies an increased risk of severe infection. Cancer patients...
Neutropenia is a condition identified by an abnormally low number of neutrophils in the bloodstream and signifies an increased risk of severe infection. Cancer patients are particularly susceptible to this condition, which can be disruptive to their treatment and even life-threatening in severe cases. Thus, it is critical to routinely monitor neutrophil counts in cancer patients. However, the standard of care to assess neutropenia, the complete blood count (CBC), requires expensive and complex equipment, as well as cumbersome procedures, which precludes easy or timely access to critical hematological information, namely neutrophil counts. Here we present a simple, low-cost, fast, and robust technique to detect and grade neutropenia based on label-free multi-spectral deep-UV microscopy. Results show that the developed framework for automated segmentation and classification of live, unstained blood cells in a smear accurately differentiates patients with moderate and severe neutropenia from healthy samples in minutes. This work has significant implications towards the development of a low-cost and easy-to-use point-of-care device for tracking neutrophil counts, which can not only improve the quality of life and treatment-outcomes of many patients but can also be lifesaving.
PubMed: 34745725
DOI: 10.1364/BOE.434465 -
Molecules (Basel, Switzerland) Jul 2023Natural polysaccharides are macromolecular substances with a wide range of biological activities. The structural modification of polysaccharides by chemical means can... (Review)
Review
Natural polysaccharides are macromolecular substances with a wide range of biological activities. The structural modification of polysaccharides by chemical means can enhance their biological activity. This paper reviews the latest research reports on the chemical modification of natural polysaccharides. At present, the modification methods of polysaccharides mainly include sulfation, phosphorylation, carboxymethylation, socialization, methylation and acetylation. The chemical and physical structures of the modified polysaccharides were detected via ultraviolet spectroscopy, FT-IR, high-performance liquid chromatography, ultraviolet spectroscopy, gas chromatography-mass spectrometry, nuclear magnetic resonance and scanning electron microscopy. Modern pharmacological studies have shown that the modified polysaccharide has various biological activities, such as antioxidant, antitumor, immune regulation, antiviral, antibacterial and anticoagulant functions in vitro. This review provides fresh ideas for the research and application of polysaccharide structure modification.
Topics: Spectroscopy, Fourier Transform Infrared; Polysaccharides; Antioxidants; Magnetic Resonance Spectroscopy; Phosphorylation
PubMed: 37513287
DOI: 10.3390/molecules28145416 -
Nature Biomedical Engineering Dec 2017Histological examination of tissues is central to the diagnosis and management of neoplasms and many other diseases and is a foundational technique for preclinical and...
Histological examination of tissues is central to the diagnosis and management of neoplasms and many other diseases and is a foundational technique for preclinical and basic research. However, commonly used bright-field microscopy requires prior preparation of micrometre-thick tissue sections mounted on glass slides-a process that can require hours or days, contributes to cost and delays access to critical information. Here, we introduce a simple, non-destructive slide-free technique that, within minutes, provides high-resolution diagnostic histological images resembling those obtained from conventional haematoxylin and eosin histology. The approach, which we named microscopy with ultraviolet surface excitation (MUSE), can also generate shape and colour-contrast information. MUSE relies on ~280 nm ultraviolet light to restrict the excitation of conventional fluorescent stains to tissue surfaces and it has no significant effects on downstream molecular assays (including fluorescence in situ hybridization and RNA sequencing). MUSE promises to improve the speed and efficiency of patient care in both state-of-the-art and low-resource settings and to provide opportunities for rapid histology in research.
Topics: Animals; Carcinoma; Histological Techniques; Humans; Image Processing, Computer-Assisted; Microscopy, Ultraviolet; Molecular Diagnostic Techniques; Pathology; Reproducibility of Results; Ultraviolet Rays
PubMed: 31015706
DOI: 10.1038/s41551-017-0165-y -
Light, Science & Applications Apr 2022Microscopy with extreme ultraviolet (EUV) radiation holds promise for high-resolution imaging with excellent material contrast, due to the short wavelength and numerous...
Microscopy with extreme ultraviolet (EUV) radiation holds promise for high-resolution imaging with excellent material contrast, due to the short wavelength and numerous element-specific absorption edges available in this spectral range. At the same time, EUV radiation has significantly larger penetration depths than electrons. It thus enables a nano-scale view into complex three-dimensional structures that are important for material science, semiconductor metrology, and next-generation nano-devices. Here, we present high-resolution and material-specific microscopy at 13.5 nm wavelength. We combine a highly stable, high photon-flux, table-top EUV source with an interferometrically stabilized ptychography setup. By utilizing structured EUV illumination, we overcome the limitations of conventional EUV focusing optics and demonstrate high-resolution microscopy at a half-pitch lateral resolution of 16 nm. Moreover, we propose mixed-state orthogonal probe relaxation ptychography, enabling robust phase-contrast imaging over wide fields of view and long acquisition times. In this way, the complex transmission of an integrated circuit is precisely reconstructed, allowing for the classification of the material composition of mesoscopic semiconductor systems.
PubMed: 35487910
DOI: 10.1038/s41377-022-00797-6 -
Biomedical Optics Express Jan 2020Immunohistochemical techniques, such as immunofluorescence (IF) staining, enable microscopic imaging of local protein expression within tissue samples. Molecular...
Immunohistochemical techniques, such as immunofluorescence (IF) staining, enable microscopic imaging of local protein expression within tissue samples. Molecular profiling enabled by IF is critical to understanding pathogenesis and is often involved in complex diagnoses. A recent innovation, known as (MUSE), uses deep ultraviolet (≈280 nm) illumination to excite labels at the tissue surface, providing equivalent images without fixation, embedding, and sectioning. However, MUSE has not yet been integrated into traditional IF pipelines. This limits its application in more complex diagnoses that rely on protein-specific markers. This paper aims to broaden the applicability of MUSE to multiplex immunohistochemistry using quantum dot nanoparticles. We demonstrate the advantages of quantum dot labels for protein-specific MUSE imaging on both paraffin-embedded and intact tissue, significantly expanding MUSE applicability to protein-specific applications. Furthermore, with recent innovations in three-dimensional ultraviolet fluorescence microscopy, this opens the door to three-dimensional IF imaging with quantum dots using ultraviolet excitation.
PubMed: 32010503
DOI: 10.1364/BOE.11.000099 -
Micromachines Mar 2023This review paper presents the recent developments in spectroelectrochemical (SEC) technologies. The coupling of spectroscopy and electrochemistry enables SEC to do a... (Review)
Review
This review paper presents the recent developments in spectroelectrochemical (SEC) technologies. The coupling of spectroscopy and electrochemistry enables SEC to do a detailed and comprehensive study of the electron transfer kinetics and vibrational spectroscopic fingerprint of analytes during electrochemical reactions. Though SEC is a promising technique, the usage of SEC techniques is still limited. Therefore, enough publicity for SEC is required, considering the promising potential in the analysis fields. Unlike previously published review papers primarily focused on the relatively frequently used SEC techniques (ultraviolet-visible SEC and surface-enhanced Raman spectroscopy SEC), the two not-frequently used but promising techniques (nuclear magnetic resonance SEC and dark-field microscopy SEC) have also been studied in detail. This review paper not only focuses on the applications of each SEC method but also details their primary working mechanism. In short, this paper summarizes each SEC technique's working principles, current applications, challenges encountered, and future development directions. In addition, each SEC technique's applicative research directions are detailed and compared in this review work. Furthermore, integrating SEC techniques into microfluidics is becoming a trend in minimized analysis devices. Therefore, the usage of SEC techniques in microfluidics is discussed.
PubMed: 36985074
DOI: 10.3390/mi14030667 -
Photoacoustics Mar 2022Ultraviolet photoacoustic microscopy (UV-PAM) has been investigated to provide label-free and registration-free volumetric histological images for whole organs, offering...
Ultraviolet photoacoustic microscopy (UV-PAM) has been investigated to provide label-free and registration-free volumetric histological images for whole organs, offering new insights into complex biological organs. However, because of the high UV absorption of lipids and pigments in tissue, UV-PAM suffers from low image contrast and shallow image depth, hindering its capability for revealing various microstructures in organs. To improve the UV-PAM imaging contrast and imaging depth, here we propose to implement a state-of-the-art optical clearing technique, CUBIC (clear, unobstructed brain/body imaging cocktails and computational analysis), to wash out the lipids and pigments from tissues. Our results show that the UV-PAM imaging contrast and quality can be significantly improved after tissue clearing. With the cleared tissue, multilayers of cell nuclei can also be extracted from time-resolved PA signals. Tissue clearing-enhanced UV-PAM can provide fine details for organ imaging.
PubMed: 34804794
DOI: 10.1016/j.pacs.2021.100313