Corresponding author BS has indicated that the images used in Fig 4C of 1 are separately captured images of the same harvested Matrigel section as shown in Fig 5Ea and 5Eb of 2, with different ...magnifications. * In Fig 4B of 1, the lower right-hand Matrigel image panel with scale 10μm (described in the caption as human dermal-derived fibroblasts) is incorrect and is a higher magnification image of the same tissue shown in the BM “MSCs” Matrigel image panel in Fig 5Fb of 2. * A region on the right-hand side of the hSpectrin Laminin Dapi panel of Fig 5D of 1 reported to show myofibers of SCID/beige/CTX (cardiotoxin) injured mice, when flipped vertically and rotated appears similar to part of the lower right-hand panel of Fig 5D of 2 reported to show myofibers from SCID/mdx mice. In response to these concerns, corresponding author BS indicated that the study reported in 2 was a preliminary approach to questions around non-canonical myogenic progenitors and that the PLOS ONE article 1 addressed further pending questions, offering advances including: * A direct comparison of the explant culture vs CD-146+-enriched populations, in terms of myogenic potential, frequency of myogenic cells and expression of markers; and * The finding that in vivo transplanted human muscle-derived CD146+ multi-clonal cell strain immunoreactivity of CD146 was restricted to microvascular walls of the interstitial tissue. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. * There are also several references to “Data not shown” in support of statements in the originally published article, which is not permitted under PLOS ONE’s editorial policies.
The Nemo's Gardensup.® project is an alternative production system for areas with scarce cultivable land but significant presence of water; thus, it is an interesting intervention to address the ...climate crisis. This work aimed to evaluate the micromorphological, biochemical, and phytochemical characteristics of Stevia rebaudiana (Bertoni) Bertoni grown underwater compared to the terrestrial specimens. The micromorphological analyses, performed on the leaves using light microscopy, fluorescence microscopy, and scanning electron microscopy, evidenced a general uniformity of the trichome morphotype and distribution pattern. The histochemical investigation indicated the simultaneous presence of terpenes and polyphenols in the trichome secreted material from the underwater samples and a prevailing polyphenolic content in the terrestrial specimens; this was also confirmed by biochemical analyses (26.6 mg GAE/g DW). The characterization of non-volatile components, performed using HPLC-MS, showed similar chemical profiles in all the samples, which were characterized by phenolic compounds and steviol glycosides. The volatile compounds, evaluated using HS-SPME coupled with GC-MS, showed sesquiterpene hydrocarbons as the main class in all the analyzed samples (80.1-93.9%). However, the control plants were characterized by a higher content of monoterpene hydrocarbons (12.1%). The underwater biosphere environment did not alter S. rebaudiana micro-morphological characters, although slight qualitative changes were evidenced for the compounds produced as a response to the growth conditions.
Fibroblasts are among the most common cell types in the stroma responsible for creating and maintaining the structural organization of the extracellular matrix in the dermis, skin regeneration, and a ...range of immune responses. Until now, the processes of fibroblast adaptation and functioning in a varying environment have not been fully understood. Modern laser microscopes are capable of studying fibroblasts in vitro and ex vivo. One-photon- and two-photon-excited fluorescence microscopy, Raman spectroscopy/microspectroscopy are well-suited noninvasive optical methods for fibroblast imaging in vitro and ex vivo. In vivo staining-free fibroblast imaging is not still implemented. The exception is fibroblast imaging in tattooed skin. Although in vivo noninvasive staining-free imaging of fibroblasts in the skin has not yet been implemented, it is expected in the future. This review summarizes the state-of-the-art in fibroblast visualization using optical methods and discusses the advantages, limitations, and prospects for future noninvasive imaging.
A series of dithienothiophene S,S-dioxide (DTTDO) dyes was designed, synthesized, and investigated for their suitability in fluorescent cell imaging. Synthetized (D-π-A-π-D)-type DTTDO derivatives ...have molecule lengths close to the thickness of the phospholipid membrane, and they contain on both ends two positively charged or neutral polar groups to increase their solubility in water and to ensure simultaneous interaction with polar groups of the inner and outer part of the cellular membrane. DTTDO derivatives exhibit absorbance and emission maxima in the 517-538 nm and 622-694 nm range, respectively, and a large Stokes shift up to 174 nm. Fluorescence microscopy experiments revealed that these compounds selectively intercalate into cell membranes. Moreover, a cytotoxicity assay conducted on a model human live cells indicates low toxicity of these compounds at the concentrations required for effective staining. With suitable optical properties, low cytotoxicity, and high selectivity against cellular structures, DTTDO derivatives are proven to be attractive dyes for fluorescence-based bioimaging.
The autofluorescence phenomenon is an inherent characteristic of lignified cells. However, in the case of Lophira alata (L. alata), the autofluorescence is nearly imperceptible during occasional ...fluorescence observations. The aim of this study is to investigate the mechanism behind the quenching of lignin’s autofluorescence in L. alata by conducting associated experiments. Notably, the autofluorescence image of L. alata observed using optical microscopy appears to be quite indistinct. Abundant extractives are found in the longitudinal parenchyma, fibers, and vessels of L. alata. Remarkably, when subjected to a benzene–alcohol extraction treatment, the autofluorescence of L. alata becomes progressively enhanced under a fluorescence microscope. Additionally, UV–Vis absorption spectra demonstrate that the extractives derived from L. alata exhibit strong light absorption within the wavelength range of 200–500 nm. This suggests that the abundant extractives in L. alata are probably responsible for the autofluorescence quenching observed in the cell walls. Moreover, the presence and quantity of these extractives have a significant impact on the fluorescence intensity of lignin in wood, resulting in a significant decrease therein. In future studies, it would be interesting to explore the role of complex compounds such as polyphenols or terpenoids, which are present in the abundant extractives, in interfering with the fluorescence quenching of lignin in L. alata.
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In the year of 2019, we celebrate the centenary of the publication of the remarkable paper by Otto Stern and Max Volmer concerning the kinetics analysis of fluorescence quenching. ...Their achievement changed enormously many fields of science with great impact in the studies of electronic spectroscopy and kinetics of organic, inorganic and biological compounds in condensed phase. The development of molecular photochemistry was in great part based on the use of the Stern-Volmer (SV) approach to investigate bimolecular interaction in the electronic excited-state. This paper reviews and summarizes the assumptions behind the Stern-Volmer equation and the extensions of the theory to other important situations. Discussions about the use of the SV approach to investigate probe and quencher distribution, association, diffusion and reaction at the molecular level obtained from advanced fluorescence microscopy methods are also included.
Phototoxicity frequently occurs during live fluorescence microscopy, and its consequences are often underestimated. Damage to cellular macromolecules upon excitation light illumination can impair ...sample physiology, and even lead to sample death. In this review, we explain how phototoxicity influences live samples, and we highlight that, besides the obvious effects of phototoxicity, there are often subtler consequences of illumination that are imperceptible when only the morphology of samples is examined. Such less apparent manifestations of phototoxicity are equally problematic, and can change the conclusions drawn from an experiment. Thus, limiting phototoxicity is a prerequisite for obtaining reproducible quantitative data on biological processes. We present strategies to reduce phototoxicity, e.g. limiting the illumination to the focal plane and suggest controls for phototoxicity effects. Overall, we argue that phototoxicity needs increased attention from researchers when designing experiments, and when evaluating research findings.
Phototoxicity during live fluorescence microscopy is a frequent side effect of excess illumination that can influence data quality. Samples can be affected by phototoxicity even before it becomes obvious from their morphology. In this review, we explore how to mitigate and control for phototoxicity to collect reliable quantitative imaging data.
Friction is generated where solids touch, thus quantitative understanding of the microscopic contact area is crucial to predict macroscopic friction. All surfaces are rough, and typically small-scale ...roughness rather than macroscopic geometry sets the contact area. Rough contact theories have been developed that predict the contact area, using the surface topography and the mechanical properties of the solids as input. However, the validity of these theories remains under debate. Systematic comparison between theory and experiment has been lacking, due to the experimental difficulty to access the contact area, especially at small length scales where idealised assumptions of contact models may not hold. Here, we use a state-of-the-art fluorescence microscopy technique to directly access the contact area of a glass-glass contact with nanometric out-of-plane resolution. We systematically vary the roughness of the contact and carefully measure the surface topography across length scales. Comparison of high-resolution contact experiments to different contact theories enables us to unambigously conclude that only elastic Persson theory quantitatively describes experimental observations.
Mycobacterium tuberculosis (Mtb) infection is initiated by inhalation of bacteria into lung alveoli, where they are phagocytosed by resident macrophages. Intracellular Mtb replication induces the ...death of the infected macrophages and the release of bacterial aggregates. Here, we show that these aggregates can evade phagocytosis by killing macrophages in a contact‐dependent but uptake‐independent manner. We use time‐lapse fluorescence microscopy to show that contact with extracellular Mtb aggregates triggers macrophage plasma membrane perturbation, cytosolic calcium accumulation, and pyroptotic cell death. These effects depend on the Mtb ESX‐1 secretion system, however, this system alone cannot induce calcium accumulation and macrophage death in the absence of the Mtb surface‐exposed lipid phthiocerol dimycocerosate. Unexpectedly, we found that blocking ESX‐1‐mediated secretion of the EsxA/EsxB virulence factors does not eliminate the uptake‐independent killing of macrophages and that the 50‐kDa isoform of the ESX‐1‐secreted protein EspB can mediate killing in the absence of EsxA/EsxB secretion. Treatment with an ESX‐1 inhibitor reduces uptake‐independent killing of macrophages by Mtb aggregates, suggesting that novel therapies targeting this anti‐phagocytic mechanism could prevent the propagation of extracellular bacteria within the lung.
Synopsis
The uptake of Mycobacterium tuberculosis (Mtb) by lung macrophages is followed by intracellular replication, cell death, and release of Mtb aggregates. This study shows that extracellular Mtb aggregates can also induce macrophage death in a contact‐dependent but uptake‐independent manner.
Contact with extracellular Mtb aggregates induces plasma membrane perturbation, calcium accumulation, and pyroptotic cell death in macrophages.
These events are driven by the Mtb type VII secretion system ESX‐1 and by the surface‐exposed lipid PDIM.
In the absence of ESX‐1 effector EsxA/EsxB secretion, the uptake‐independent killing is mediated by the ESX‐1 secreted protein EspB.
Treatment with a small molecule that inhibits ESX‐1‐mediated secretion reduces uptake‐independent killing of macrophages by Mtb aggregates.
Mycobacterium tuberculosis aggregates evade phagocytosis by inducing macrophage pyroptosis via a mechanism involving its type VII secretion system ESX‐1 and surface lipid PDIM.
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