Correction of chromatic shift is necessary for precise registration of multicolor fluorescence images of biological specimens. New emerging technologies in fluorescence microscopy with increasing ...spatial resolution and penetration depth have prompted the need for more accurate methods to correct chromatic aberration. However, the amount of chromatic shift of the region of interest in biological samples often deviates from the theoretical prediction because of unknown dispersion in the biological samples. To measure and correct chromatic shift in biological samples, we developed a quadrisection phase correlation approach to computationally calculate translation, rotation, and magnification from reference images. Furthermore, to account for local chromatic shifts, images are split into smaller elements, for which the phase correlation between channels is measured individually and corrected accordingly. We implemented this method in an easy-to-use open-source software package, called Chromagnon, that is able to correct shifts with a 3D accuracy of approximately 15 nm. Applying this software, we quantified the level of uncertainty in chromatic shift correction, depending on the imaging modality used, and for different existing calibration methods, along with the proposed one. Finally, we provide guidelines to choose the optimal chromatic shift registration method for any given situation.
Extracellular vesicles such as exosomes are generally covered with an array of glycans, which are controlled by the host-cell glyco-synthetic machinery, similar to secreted and membrane ...glycoproteins. Several exosome subpopulations classified by their tetraspanin expression have been investigated in the context of diseases. However, a comparative analysis of their glycomics has never been attempted. Herein, we report a method for the comparative glycomic analysis of exosome subpopulations among pancreatic cancer cell lines. Glycomic profiles were obtained for extracellular vesicles, secreted glycoproteins, and membrane glycoproteins from eight cell lines. Statistical analyses revealed high populations of PHA-L-binding proteins in the vesicles. The surfaces of extracellular vesicles were labeled with Cy3 and captured by magnetic beads with antibodies against tetraspanins (CD9, CD63, and CD81). The coprecipitated vesicles were lysed and subjected to a lectin microarray analysis. A hierarchical clustering analysis using 19 glycomic profiles confirmed that most subpopulations, except CD81-positive exosomes, could be distinguished according to the host-cell species. Principal component analysis and subsequent lectin-affinity capturing of intact exosomes highlighted that CD81-positive exosomes preferentially expressed not PHA-L- but LEL-binding proteins on their surfaces. These data suggested that exosomal glycomics depended on the host-cell type and subpopulation.
The structure of chromosomes dramatically changes upon entering meiosis to ensure the successful progression of meiosis-specific events. During this process, a multilayer proteinaceous structure ...called a synaptonemal complex (SC) is formed in many eukaryotes. However, in the fission yeast
Schizosaccharomyces pombe
, linear elements (LinEs), which are structures related to axial elements of the SC, form on the meiotic cohesin-based chromosome axis. The structure of LinEs has been observed using silver-stained electron micrographs or in immunofluorescence-stained spread nuclei. However, the fine structure of LinEs and their dynamics in intact living cells remain to be elucidated. In this study, we performed live cell imaging with wide-field fluorescence microscopy as well as 3D structured illumination microscopy (3D-SIM) of the core components of LinEs (Rec10, Rec25, Rec27, Mug20) and a linE-binding protein Hop1. We found that LinEs form along the chromosome axis and elongate during meiotic prophase. 3D-SIM microscopy revealed that Rec10 localized to meiotic chromosomes in the absence of other LinE proteins, but shaped into LinEs only in the presence of all three other components, the Rec25, Rec27, and Mug20. Elongation of LinEs was impaired in double-strand break-defective
rec12
−
cells. The structure of LinEs persisted after treatment with 1,6-hexanediol and showed slow fluorescence recovery from photobleaching. These results indicate that LinEs are stable structures resembling axial elements of the SC.
The biochemical transduction of excitatory synaptic signals occurs in the cytoplasm within dendritic spines. The associated reaction kinetics are shaped by the mobility of the signaling molecules; ...however, accurate monitoring of diffusional events within the femtoliter-sized spine structures has not yet been demonstrated. Here, we applied two-photon fluorescence correlation spectroscopy and raster image correlation spectroscopy to monitor protein dynamics within spines, revealing that F-actin restricts the mobility of proteins with a molecular mass of >100 kDa. This restriction is transiently removed during actin remodeling at the initial phase of spine structural plasticity. Photobleaching experiments combined with super-resolution imaging indicate that this increase in mobility facilitates molecular interactions, which may modulate the functions of key postsynaptic signaling molecules, such as Tiam1 and CaMKII. Thus, actin polymers in dendritic spines act as precise temporal regulators of molecular diffusion and modulate signal transduction during synaptic plasticity.
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•Two-photon FCS and RICS report protein dynamics within dendritic spines•Actin polymers within spines restrict molecular mobility in a size-specific manner•Actin remodeling during the initial phase of structural LTP increases molecular mobility•Actin polymers in spines act as precise temporal regulators of postsynaptic signaling
Obashi et al. show that actin polymers within dendritic spines restrict mobility of large molecules using optical measurements of fluorescence correlation. Acute actin remodeling induced by plasticity-inducing stimuli increases the mobility of large postsynaptic signaling molecules, which regulate long-term changes in synaptic property.
Roadmap of incoherent digital holography Tahara, Tatsuki; Zhang, Yaping; Rosen, Joseph ...
Applied physics. B, Lasers and optics,
11/2022, Volume:
128, Issue:
11
Journal Article
Peer reviewed
Open access
This roadmap article focuses on spatially incoherent digital holography (IDH). Representative IDH methods such as optical scanning holography (OSH), Fresnel incoherent correlation holography (FINCH), ...coded aperture correlation holography (COACH), IDH with a Fresnel zone aperture, and IDH with an interferometer along with a state-of-the-art optical device are introduced as modern IDH methods. We describe these IDH techniques with applications of three-dimensional (3D) imagers, 3D thermography, and 3D microscopy.
3D structured illumination microscopy (3D-SIM) is the super-resolution technique of choice for multicolor volumetric imaging. Here we provide a validated sample preparation protocol for labeling ...nuclei of cultured mammalian cells, image acquisition and registration practices, and downstream image analysis of nuclear structures and epigenetic marks. Using immunostaining and replication labeling combined with image segmentation, centroid mapping and nearest-neighbor analyses in open-source environments, 3D maps of nuclear structures are analyzed in individual cells and normalized to fluorescence standards on the nanometer scale. This protocol fills an unmet need for the application of 3D-SIM to the technically challenging nuclear environment, and subsequent quantitative analysis of 3D nuclear structures and epigenetic modifications. In addition, it establishes practical guidelines and open-source solutions using ImageJ/Fiji and the TANGO plugin for high-quality and routinely comparable data generation in immunostaining experiments that apply across model systems. From sample preparation through image analysis, the protocol can be executed within one week.
High-mobility group box 1 (HMGB1) is a DNA-binding protein abundantly expressed in the nucleus that has gained much attention for its regulation of immunity and inflammation. Despite this, whether ...and how HMGB1 contributes to protective and/or pathological responses in vivo is unclear. In this study, we constructed Hmgb1 -floxed (Hmgb1 ᶠ/ᶠ) mice to achieve the conditional inactivation of the gene in a cell- and tissue-specific manner by crossing these mice with an appropriate Cre recombinase transgenic strain. Interestingly, although mice with HMGB1 ablation in myeloid cells apparently develop normally, they are more sensitive to endotoxin shock compared with control mice, which is accompanied by massive macrophage cell death. Furthermore, these mice also show an increased sensitivity to Listeria monocytogenes infection. We also provide evidence that the loss of HMGB1 in macrophages results in the suppression of autophagy, which is commonly induced by lipopolysaccharide stimulation or L. monocytogenes infection. Thus, intracellular HMGB1 contributes to the protection of mice from endotoxemia and bacterial infection by mediating autophagy in macrophages. These newly generated HMGB1 conditional knockout mice will serve a useful tool with which to study further the in vivo role of this protein in various pathological conditions.
Fluorescence microscopy allows us to observe fluorescently labeled molecules in diverse biological processes and organelle structures within living cells. However, the diffraction limit restricts its ...spatial resolution to about half of its wavelength, limiting the capability of biological observation at the molecular level. Structured-illumination microscopy (SIM), a type of super-resolution microscopy, doubles the spatial resolution in all three dimensions by illuminating the sample with a patterned excitation light, followed by computer reconstruction. SIM uses a relatively low illumination power compared with other methods of super-resolution microscopy and is easily available for multicolor imaging. SIM has great potential for meeting the requirements of live-cell imaging. Recent developments in diverse types of SIM have achieved higher spatial (∼50 nm lateral) and temporal (∼100 Hz) resolutions. Here, we review recent advancements in SIM and discuss its application in noninvasive live-cell imaging.
The synthesis of several meso-triaryl-subporphyrins, based on utilizing tripyrrolylborane as a precursor in a reaction with arylaldehydes (where aryl = phenyl, 4- and 3-pyridyl, tolyl, ...4-methoxyphenyl, and 4-(trifluoromethyl)phenyl) under reflux in propionic acid, is reported. All of the compounds have been successfully characterized by 1H−, 13C−, and 13C−1H 2D NMR, electronic absorption, magnetic circular dichroism (MCD), IR, and fluorescence spectroscopy, together with cyclic (CV) and differential pulse (DPV) voltammetry. The X-ray structure of tris{(trifluoromethyl)phenyl}subporphyrin was found to be slightly domed and similar to that of the recently reported hexaethylsubtriazaporphyrin (Inorg. Chem. 2006, 45, 6148). The electronic absorption spectra of all of the subporphyrins contain intense Soret bands in the 370−380 nm region and weaker Q bands in the 420−550 nm region, which are at shorter wavelengths than those observed (at ca. 400−420 nm and ca. 450−650 nm, respectively) for tetrapyrrole porphyrins. The intensity of the Q00 band decreases as the meso-aryl groups become more electron withdrawing. These characteristics can be rationalized by using Gouterman's four-orbital model as a conceptual framework. The MCD bands observed in the Q band region of the subporphyrins (subPs) spectra consistently show a −ve/+ve intensity pattern in ascending energy, while, in contrast, the sign sequence of the bands observed in the Soret band region change dramatically depending on the nature of the aryl groups: from +ve/−ve in the case of the electron withdrawing 4-pyridyl, 4-(trifluoromethyl)phenyl and 3-pyridyl groups to −ve only for phenyl and −ve/+ve for the electron donating 4-tolyl and 4-methoxyphenyl groups. S1 fluorescence emission was observed in the 490−620 nm region. The quantum yields (φF) in benzene (φF = 0.10−0.12) are similar to that of metal-free tetraphenylporphyrin (H2TPP) (φF = 0.11) but are somewhat lower in the case of ethanol (φF = 0.06−0.07) due to the higher polarity. The redox potential differences observed between the first oxidation and reduction steps are in the 2.52−2.64 V range, which is larger than that of normal porphyrins (ca. 2.25 V). Molecular orbital (MO) calculations of these compounds help to provide an enhanced understanding of the spectroscopic and electrochemical properties. A byproduct of the synthesis was characterized using X-ray crystallography and a range of spectroscopic techniques. A subporphyrin μ-oxo dimer was prepared and characterized.
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