Optical Fluorescence Microscopy Research Articles

Illuminating the nanoscopic world of viruses by fluorescence super-resolution microscopy

The recent revolution in optical fluorescence microscopy, supported by the optimization of both spatial resolution and acquisition speed, led to the ability to visualize nano-scaled objects. Currently, the use of a new generation of super-resolution fluorescence microscopes coupled to improved fluorescent probes gives the possibility to study the replicative cycle of viruses in living cells, at the single-virus and molecule level. In this review, after a brief chronological description of these new approaches, we highlight several examples of super-resolution microscopies that have allowed to revisit our understanding of several human viruses and of host-pathogen interactions.

Illuminating the nanoscopic world of viruses by fluorescence super-resolution microscopy.

The recent revolution in optical fluorescence microscopy, supported by the optimization of both spatial resolution and acquisition speed, led to the ability to visualize nano-scaled objects. Currently, the use of a new generation of super-resolution fluorescence microscopes coupled to improved fluorescent probes gives the possibility to study the replicative cycle of viruses in living cells, at the single-virus and molecule level. In this review, after a brief chronological description of these new approaches, we highlight several examples of super-resolution microscopies that have allowed to revisit our understanding of several human viruses and of host-pathogen interactions.

Correlative nanoscopy: A multimodal approach to molecular resolution.

Atomic force microscopy (AFM) is a nano‐mechanical tool uniquely suited for biological studies at the molecular scale. AFM operation is based on mechanical interaction between the tip and the sample, a mechanism of contrast capable of measuring different information, including surface topography, mechanical, and electrical properties. However, the lack of specificity highlights the need to integrate AFM data with other techniques providing compositional hints. In particular, optical microscopes based on fluorescence as a mechanism of contrast can access the local distribution of specific molecular species. The coupling between AFM and super‐resolved fluorescence microscopy solves the resolution mismatch between AFM and conventional fluorescence optical microscopy. Recent advances showed that also the inherently label‐free imaging capabilities of the AFM are fundamental to complement the fluorescence images. In this review, we have presented a brief historical view on correlative microscopy, and, finally, we have summarized the progress of correlative AFM‐super‐resolution microscopy in biological research.

Maskless Preparation of Spatially‐Resolved Plasmonic Nanoparticles on Polydimethylsiloxane via In Situ Fluoride‐Assisted Synthesis

AbstractHere, a fluoride‐assisted route for the controlled in‐situ synthesis of metal nanoparticles (NPs) (i.e., AgNPs, AuNPs) on polydimethylsiloxane (PDMS) is reported. The size and coverage of the NPs on the PDMS surface are modulated with time and over space during the synthetic process, leveraging the improved yield (10×) and faster kinetics (100×) of NP formation in the presence of F−ions, compared to fluoride‐free approaches. This enables the maskless preparation of both linear and step gradients and patterns of NPs in 1D and 2D on the PDMS surface. As an application in flexible plasmonics/photonics, continuous and step‐wise spatial modulations of the plasmonic features of PDMS slabs with 1D and 2D AgNP gradients on the surface are demonstrated. An excellent spatially resolved tuning of key optical parameters, namely, optical density from zero to 5 and extinction ratio up to 100 dB, is achieved with AgNP gradients prepared in AgF solution for 12 minutes; the performance are comparable to those of commercial dielectric/interference filters. When used as a rejection filter in optical fluorescence microscopy, the AgNP‐PDMS slabs are able to reject the excitation laser at 405 nm and retain the green fluorescence of microbeads (100 µm) used as test cases.

Oral dysbiosis exacerbates the virulence of Candida parapsilosis sensu stricto via up-regulation of the CPH2 biofilm master gene

Candida parapsilosis sensu stricto is the second to third most frequent cause of candidemia. Studies place this yeast as a frequent colonizer of niches of the oral cavity, predominantly in pathological conditions. We hypothesize that a buccal environment in dysbiosis enhances the virulence of C. parapsilosis sensu stricto. Objective: To evaluate at the phenotype and molecular level the production of biofilm in oral isolates of C. parapsilosis sensu stricto and correlate the results with the clinical origin (dysbiosis versus eubiosis). Material and methods: The biofilm-forming ability was compared in 50 oral isolates of C. parapsilosis sensu stricto obtained from patients with and without oral dysbiosis; by quantification of biofilm biomass and metabolic activity. The results were corroborated by optical and confocal fluorescence microscopy, and correlated with the transcriptional activity of CPH2, by RT-qPCR. The data were analyzed by Excel 2010, and InfoStat 2018, with a 95% confidence interval. Results: The metabolic activity in biofilm was significantly higher in oral dysbiosis relative to control (p = 0.0025). Basal expression of CPH2 increased 2.8 times more in oral dysbiosis related to the control condition and showed no significant differences with pathogenic isolates of this same yeast, derived from onychomycosis lesions. Conclusion: The oral cavity in dysbiosis increases the virulence of C. parapsilosis sensu stricto due to possible changes in epigenetic marks. This finding suggests that the oral cavity in dysbiosis may be an alternative route to the skin in the epidemiology of nosocomial candidemia.

Axial line-scanning stimulated emission depletion fluorescence correlation spectroscopy.

Investigating the dynamics and interactions of biomolecules within or attached to membranes of living cells is crucial for understanding biology at the molecular level. In this pursuit, classical, diffraction-limited optical fluorescence microscopy is widely used, but it faces limitations due to (1) the heterogeneity of biomembranes on the nanoscale and (2) the intrinsic motion of membranes with respect to the focus. Here we introduce a new confocal microscopy-based fluctuation spectroscopy technique aimed at alleviating these two problems, called axial line-scanning stimulated emission depletion fluorescence correlation spectroscopy (axial ls-STED-FCS). Axial line scanning by means of a tunable acoustic gradient index of refraction lens provides a time resolution of a few microseconds, which is more than two orders of magnitude greater than that of conventional, lateral line-scanning fluorescence correlation spectroscopy (typically around 1 ms). Using STED excitation, the observation area on the membrane can be reduced 10-100 fold, resulting in sub-diffraction spatial resolution and the ability to study samples with densely labeled membranes. Due to these attractive properties, we expect that the axial ls-STED-FCS will find wide application, especially in the biomolecular sciences.

Microscopy of Diffuse Nematic–Isotropic Transition in Main-Chain Nematic Liquid-Crystal Elastomers

Nematic–isotropic phase transition in liquid-crystal elastomers (LCEs), which drastically alters the macroscopic properties of the material, is attractive for both fundamental physics and applications. Herein, we introduce new experimental methods based on polarized and fluorescence optical microscopy to characterize the phase transition of polydomain LCEs, monitoring the order parameter and the nematic domain structure. It is demonstrated that polydomain main-chain nematic LCEs exhibit an ordering transition very different from the classical first-order one, having a diffuse continuous nature. Unexpectedly, the characteristic correlation length of the fading polydomain structure remains unchanged across the transition.

An Optically Reconfigurable Förster Resonance Energy Transfer Process for Broadband Switchable Organic Single-Mode Microlasers

An Optically Reconfigurable Förster Resonance Energy Transfer Process for Broadband Switchable Organic Single-Mode Microlasers

Direct visualization of electrochemical reactions and heterogeneous transport within porous electrodes in operando by fluorescence microscopy

Optical fluorescence microscopy is shown to enable both high spatial and temporal resolution of redox-dependent fluorescence in flowing electrolytes. We report the use of fluorescence microscopy coupled with electrochemistry to directly observe the reaction and transport of redox-active quinones within porous carbon electrodes in operando. We observe surprising electrolyte channeling features within several porous electrodes, leading to spatially distinguishable advection-dominated and diffusion-dominated regions. These results challenge the common assumption that transport in porous electrodes can be approximated by a homogeneous Darcy-like permeability, particularly at the length scales relevant to many electrochemical systems such as redox flow batteries. This work presents a new platform to provide highly resolved spatial and temporal insight into electrolyte reactions and transport behavior within porous electrodes.

Interplay Between Nanoplastics and the Immune System of the Mediterranean Sea Urchin Paracentrotus lividus

The present study highlights for the first time the interplay between model nanoplastics, such as the carboxyl-modified polystyrene nanoparticles (PS-COOH, 60 nm) NPs and the coelomocytes of the sea urchin Paracentrotus lividus, a benthic grazer widely distributed in Mediterranean coastal area, upon acute in vitro exposure (4 h) (5 and 25 μg mL–1). Insight into PS-COOH trafficking (uptake and clearance) and effects on immune cell functions (i.e., cell viability, lysosomal membrane stability, and phagocytosis) are provided. Dynamic Light Scattering analysis reveals that PS NP suspensions in CF undergo a quick agglomeration, more pronounced for PS-COOH (608.3 ± 43 nm) compared to PS-NH2 (329.2 ± 5 nm). However, both PS NPs are still found as nano-scale agglomerates in CF after 4 h of exposure, as shown by the polydispersity index > 0.3 associated with the presence of different PS NP size populations in the CF. The observed changes in ζ-potential upon suspension in CF (–11.1 ± 3 mV and –12.1 ± 4 mV for PS-COOH and PS-NH2, respectively) confirm the formation of a bio-corona on both PS NPs. Optical fluorescence microscopy and fluorimetric analyses using fluorescently labeled PS-COOH (60 nm) reveal a fast uptake of PS-COOH primarily by phagocytes within 1 h of exposure. Upon transfer to PS NP-free CF, a significant decrease in fluorescence signal is observed, suggesting a fast cell clearance. No effect on cell viability is observed after 4 h of exposure to PS-COOH, however a significant decrease in lysosomal membrane stability (23.7 ± 4.8%) and phagocytic capacity (63.43 ± 3.4%) is observed at the highest concentration tested. Similarly, a significant reduction in cell viability, lysosomal membrane stability and phagocytosis is found upon exposure to PS-NH2 (25 μg mL–1), which confirms the important role of surface charges in triggering immunotoxicity. Overall, our results show that, although being quickly internalized, PS-COOH can be easily eliminated by the coelomocytes but may still be able to trigger an immune response upon long-term exposure scenarios. Taking into account that sediments along Mediterranean coasts are a sink for micro- and nanoplastics, the latter can reach concentrations able to exceed toxicity-thresholds for marine benthic species.

Improving the Electrical Percolating Network of Carbonaceous Slurries by Superconcentrated Electrolytes: An Electrochemical Impedance Spectroscopy Study.

Semisolid redox flow batteries simultaneously address the need for high energy density and design flexibility. The electrical percolating network and electrochemical stability of the flowable electrodes are key features that are required to fully exploit the chemistry of the semisolid slurries. Superconcentrated electrolytes are getting much attention for their wide electrochemical stability window that can be exploited to design high-voltage batteries. Here, we report on the effect of the ion concentration of superconcentrated electrolytes on the electronic percolating network of carbonaceous slurries. Slurries based on different concentrations of lithium bis(trifluoromethane)sulfonamide in tetraethylene glycol dimethyl ether (0.5, 3, and 5 mol/kg) at different content of Pureblack carbon (from 2 up to 12 wt %) have been investigated. The study was carried out by coupling electrochemical impedance spectroscopy (EIS), optical fluorescence microscopy, and rheological measurements. A model that describes the complexity and heterogeneity of the semisolid fluids by multiple conductive branches is also proposed. For the first time, to the best of our knowledge, we demonstrate that besides their recognized high electrochemical stability, superconcentrated electrolytes enable more stable and electronically conductive slurry. Indeed, the high ionic strength of the superconcentrated solution shields interparticle interactions and enables better carbon dispersion and connections.

Mesoscopic Optical Imaging of the Pancreas-Revisiting Pancreatic Anatomy and Pathophysiology.

The exocrine-endocrine multipart organization of the pancreas makes it an exceedingly challenging organ to analyze, quantitatively and spatially. Both in rodents and humans, estimates of the pancreatic cellular composition, including beta-cell mass, has been largely relying on the extrapolation of 2D stereological data originating from limited sample volumes. Alternatively, they have been obtained by low resolution non-invasive imaging techniques providing little detail regarding the anatomical organization of the pancreas and its cellular and/or molecular make up. In this mini-review, the state of the art and the future potential of currently existing and emerging high-resolution optical imaging techniques working in the mm-cm range with μm resolution, here referred to as mesoscopic imaging approaches, will be discussed regarding their contribution toward a better understanding of pancreatic anatomy both in normal conditions and in the diabetic setting. In particular, optical projection tomography (OPT) and light sheet fluorescence microscopy (LSFM) imaging of the pancreas and their associated tissue processing and computational analysis protocols will be discussed in the light of their current capabilities and future potential to obtain more detailed 3D-spatial, quantitative, and molecular information of the pancreas.

Influence of Carbon Primary Nano Materials in Polymer Modified Binders on the Temperature Sensitivity of Asphalt Concrete During Operation

The influence of the content of single-walled carbon nanotubes on the basic physical and mechanical properties of polymer-bitumen binders is studied. The effect of nanomodifiers on the morphostructural features of polymer-bitumen binders was studied using optical methods and fluorescence microscopy. The rational content of the modifying additive (0.0001 %) in the composition of the binder, which ensures the achievement of the specified parameters, is established. The influence of single-walled carbon nanotubes on morphostructural features is established. the nanomodifier initiates the processes of structuring interfacial layers, which leads to strengthening of physical bonds between the polymer macromolecules and the dispersed phase of the organic binder, which naturally affects the improvement of heat resistance, adhesive and cohesive strength of the binder. The study of polymer-bituminous binder films allowed us to record that when the nanomodifier is introduced, the polymer macromolecules decrease and their distribution over the binder volume is more uniform, which ensures its stability. It was found that the introduction of a rational amount of nanomodifier allows you to expand the required temperature range of asphalt concrete pavement performance by 25-34 %, while reducing the polymer content by 38 %.

Brief descriptions of the principles of prominent methods used to study the penetration of materials into human hair and a review of examples of their use.

Consumers are attracted to the latest fashion trends and different looks. This drives the search for novel hair treatments. Some chemicals present in hair treatment products can penetrate the hair shaft. These materials can either nourish or injure the hair cortex. Different techniques have been used to investigate the mechanism of molecule penetration and the conditions under which penetration occurs. This article reviews the techniques applied for this purpose. Various microscopy techniques are used to capture clear and colourful images to determine the diffusion pathways and the exact location of the molecules under study. However, the laborious sample preparation often leads to sample destruction since cross-sectioning is often required. While various other techniques have been successfully used for investigating the penetration methods, most of these require different amounts of work to be put in for sample preparation and instrumentation. Several spectroscopic techniques have been used to study the penetration of the molecules because of the high levels of accuracy and the quick response time of these techniques. Moreover, the samples are not damaged during the investigation.

Addressable graphene encapsulation of wet specimens on a chip for optical, electron, infrared and X-ray based spectromicroscopy studies.

Label-free spectromicroscopy methods offer the capability to examine complex cellular phenomena. Electron and X-ray based spectromicroscopy methods, though powerful, have been hard to implement with hydrated objects due to the vacuum incompatibility of the samples and due to the parasitic signals from (or drastic attenuation by) the liquid matrix surrounding the biological object of interest. Similarly, for many techniques that operate at ambient pressure, such as Fourier transform infrared spectromicroscopy (FTIRM), the aqueous environment imposes severe limitations due to the strong absorption of liquid water in the infrared regime. Here we propose a microfabricated multi-compartmental and reusable hydrated sample platform suitable for use with several analytical techniques, which employs the conformal encapsulation of biological specimens by a few layers of atomically thin graphene. Such an electron, X-ray, and infrared transparent, molecularly impermeable and mechanically robust enclosure preserves the hydrated environment around the object for a sufficient time to allow in situ examination of hydrated bio-objects with techniques operating under both ambient and high vacuum conditions. An additional hydration source, provided by hydrogel pads lithographically patterned in the liquid state near/around the specimen and co-encapsulated, has been added to further extend the hydration lifetime. Note that the in-liquid lithographic electron beam-induced gelation procedure allows for addressable capture and immobilization of the biological cells from the solution. Scanning electron microscopy and optical fluorescence microscopy, as well as synchrotron radiation based FTIR and X-ray fluorescence microscopy, have been used to test the applicability of the platform and for its validation with yeast, A549 human carcinoma lung cells and micropatterned gels as biological object phantoms.

Investigating molecular crowding during cell division and hyperosmotic stress in budding yeast with FRET.

Cell division, aging, and stress recovery triggers spatial reorganization of cellular components in the cytoplasm, including membrane bound organelles, with molecular changes in their compositions and structures. However, it is not clear how these events are coordinated and how they integrate with regulation of molecular crowding. We use the budding yeast Saccharomyces cerevisiae as a model system to study these questions using recent progress in optical fluorescence microscopy and crowding sensing probe technology. We used a Förster Resonance Energy Transfer (FRET) based sensor, illuminated by confocal microscopy for high throughput analyses and Slimfield microscopy for single-molecule resolution, to quantify molecular crowding. We determine crowding in response to cellular growth of both mother and daughter cells, in addition to osmotic stress, and reveal hot spots of crowding across the bud neck in the burgeoning daughter cell. This crowding might be rationalized by the packing of inherited material, like the vacuole, from mother cells. We discuss recent advances in understanding the role of crowding in cellular regulation and key current challenges and conclude by presenting our recent advances in optimizing FRET-based measurements of crowding whilst simultaneously imaging a third color, which can be used as a marker that labels organelle membranes. Our approaches can be combined with synchronised cell populations to increase experimental throughput and correlate molecular crowding information with different stages in the cell cycle.

Fluorescent proteins of the EosFP clade: intriguing marker tools with multiple photoactivation modes for advanced microscopy.

Optical fluorescence microscopy has taken center stage in the exploration of biological structure and dynamics, especially on live specimens, and super-resolution imaging methods continue to deliver exciting new insights into the molecular foundations of life. Progress in the field, however, crucially hinges on advances in fluorescent marker technology. Among these, fluorescent proteins (FPs) of the GFP family are advantageous because they are genetically encodable, so that live cells, tissues or organisms can produce these markers all by themselves. A subclass of them, photoactivatable FPs, allow for control of their fluorescence emission by light irradiation, enabling pulse-chase imaging and super-resolution microscopy. In this review, we discuss FP variants of the EosFP clade that have been optimized by amino acid sequence modification to serve as markers for various imaging techniques. In general, two different modes of photoactivation are found, reversible photoswitching between a fluorescent and a nonfluorescent state and irreversible green-to red photoconversion. First, we describe their basic structural and optical properties. We then summarize recent research aimed at elucidating the photochemical processes underlying photoactivation. Finally, we briefly introduce various advanced imaging methods facilitated by specific EosFP variants, and show some exciting sample applications.

Optical tweezer and TIRF microscopy for single molecule manipulation of RNA/DNA nanostructures including their rubbery property and single molecule counting.

Life science is often focused on the microscopic level. Single-molecule technology has been used to observe components at the micro- or nanoscale. Single-molecule imaging provides unprecedented information about the behavior of individual molecules in contrast to the information from ensemble methods that average the information of many molecules in various states. A typical feature of living systems is motion. The lack of synchronicity of motion biomachines in living systems makes it challenging to image the motion process with high resolution. Thus, single-molecule technology is especially useful for real-time study on motion mechanism of biomachines, such as viral DNA packaging motor, or other ATPases. The most common optical instrumentations in single-molecule studies are optical tweezers and single molecule total internal refection fluorescence microscopy (smTIRF). Optical tweezers are the force-based technique. The analysis of RNA using optical tweezer has led to the discovery of the rubbery or amoeba property of RNA nanoparticles for compelling vessel extravasation to enhance tumor targeting and fast renal excretion. The rubbery property of RNA lends mechanistic evidence for RNAs use as an ideal reagent in cancer treatment with undetectable toxicity. Single molecule photobleaching allows for the direct counting of biomolecules. This technique was invented for single molecule counting of RNA in the phi29 DNA packaging motor to resolve the debate between five and six copies of RNA in the motor. The technology has subsequently extended to counting components in biological machines composed of protein, DNA, and other macromolecules. In combination with statistical analysis, it reveals biomolecular mechanisms in detail and leads to the development of ultra-sensitive sensors in diagnosis and forensics. This review focuses on the applications of optical tweezers and fluorescence-based techniques as single-molecule technologies to resolve mechanistic questions related to RNA and DNA nanostructures.

Utilising fluorescence spectroscopy and optical microscopy to investigate bitumen long-term ageing

Linking the chemical composition of bitumen to its microstructure is challenge due to its complexity, viscoelasticity and sensitivity to high energy input. Therefore, fluorescence spectroscopy combined with fluorescence microscopy are promising tools, as they are capable to link the materials fluorescence behaviour to its respective microstructural features. Herein, these techniques were coupled with dark-field microscopy to gain insight on the chemical composition of these auto fluorescent microstructural features and their changes during long-term-ageing (LTA). The results show that upon LTA a loss in fluorescence intensity goes in line with a shift to higher emission wavelength. While the overall intensity decreases during ageing the density and number of fluorescent particles increases. Dark-field microscopy confirms that these fluorescent particles show particle light scattering, proving that on the micro-scale bitumen is a non-homogeneous material.

Functional-characteristics of Zanthoxylum schinifolium (Siebold & Zucc.) essential oil nanoparticles

Zanthoxylum schinifolium (Siebold & Zucc.) is a folk and popular aromatic plant due to its exceptional biological properties. However, its use is limited in the industry due to its essential oil volatility and rapid degradation. In the current work, nanotechnology was applied to optimize physicochemical characteristics, long-term stability, and antioxidant performance of essential oil isolated from Z. schinifolium (ZSEO). Chromatographic analysis of isolated sonochemical ZSEO showed that linalool (57.54 %) is the main component. A distinctive and stable ZSEO nanoemulsion (ZSEO-NE) was successfully fabricated through designing an energy-saving, efficient, and applicable approach. response surface methodology (RSM) based on Box–Behnken design was the approach used for optimization processes. Double homogenization processes were successfully applied based on the sonochemical cavitation phenomenon, together with a high-pressure micro-fluidization homogenizer to form nano-scale size droplets. Octenyl succinic modified starch (OSA-MS) was the wall material and surfactant used. Tween-80 was used as a non-ionic surfactant. refined, bleached, and deodorized sunflower oil was served as a carrier material. dynamic light scattering (DLS) technique was used to analyze the droplet hydrodynamic diameter (DHD) (nm), polydispersity index (PdI), ζ-potential (mV). Optical microscopy, Fluorescence microscopy, and confocal laser scanning microscopy (CLSM) were applied for microstructure studies of ZSEO-NE. Gravitational separation and stability of physicochemical properties during long-term storage were evaluated. The optimum values of the mean DHD, PdI, and ζ-potential of ZSEO-NE ranged from 128.8 nm, 141.0, and -44.0 mV, respectively. The findings of the RSM analysis showed considerable agreement between the predictive values and the actual values, in a clear indication that the quadratic model obtained from the BBD matrix covers the experimental range of the current study. Treatment of ZSEO-NE showed maximum stability and less susceptible to droplet aggregation without noticeable separation between aqueous and organic phases during long-term storage.