In vivo femtosecond laser nanosurgery of the cell wall enabling patch-clamp measurements on filamentous fungi.

Studying the membrane physiology of filamentous fungi is key to understanding their interactions with the environment and crucial for developing new therapeutic strategies for disease-causing pathogens. However, their plasma membrane has been inaccessible for a micron-sized patch-clamp pipette for pA current recordings due to the rigid chitinous cell wall. Here, we report the first femtosecond IR laser nanosurgery of the cell wall of the filamentous fungi, which enabled patch-clamp measurements on protoplasts released from hyphae. A reproducible and highly precise (diffraction-limited, submicron resolution) method for obtaining viable released protoplasts was developed. Protoplast release from the nanosurgery-generated incisions in the cell wall was achieved from different regions of the hyphae. The plasma membrane of the obtained protoplasts formed tight and high-resistance (GΩ) contacts with the recording pipette. The entire nanosurgical procedure followed by the patch-clamp technique could be completed in less than 1 hour. Compared to previous studies using heterologously expressed channels, this technique provides the opportunity to identify new ionic currents and to study the properties of the ion channels in the protoplasts of filamentous fungi in their native environment.

Non-fluorescent transient states of tyrosine as a basis for label-free protein conformation and interaction studies.

The amino acids tryptophan, tyrosine, and phenylalanine have been extensively used for different label-free protein studies, based on the intensity, lifetime, wavelength and/or polarization of their emitted fluorescence. Similar to most fluorescent organic molecules, these amino acids can undergo transitions into dark meta-stable states, such as triplet and photo-radical states. On the one hand, these transitions limit the fluorescence signal, but they are also highly environment-sensitive and can offer an additional set of parameters, reflecting interactions, folding states, and immediate environments around the proteins. In this work, by analyzing the average intensity of tyrosine emission under different excitation modulations with the transient state monitoring (TRAST) technique, we explored the photo physics of tyrosine as a basis for such environment-sensitive readouts. From how the dark state transitions of tyrosine varied with excitation intensity and solvent conditions we first established a photophysical model for tyrosine. Next, we studied Calmodulin (containing two tyrosines), and how its conformation is changed upon calcium binding. From these TRAST experiments, performed with 280 nm time-modulated excitation, we show that tyrosine dark state transitions clearly change with the calmodulin conformation, and may thus represent a useful source of information for (label-free) analyses of protein conformations and interactions.

An upgrade of the primary length standard of Republic of Serbia where digital stabilization is performed by Arduino Due board.

An upgrade of the electronic system for frequency stabilization of the HeNe laser, primary length standard of Republic of Serbia, based on digital electronics, is described. Arduino microcontrollers have been used for stabilization, and laptop computer has been used only to communicate with the user. In addition, an analog electronics has been developed in order to boost the performance of the setup. The setup is simple and inexpensive, made of off-the-shelf electronics components. Despite this, good performances have been achieved.

Interactions of ultrashort laser pulses with hemoglobin: Photophysical aspects and potential applications.

Hemoglobin (Hb), a life-sustaining and highly abundant erythrocyte protein, is not readily fluorescent. A few studies have already reported Two-Photon Excited Fluorescence (TPEF) of Hb, however, the mechanisms through which Hb becomes fluorescent upon interaction with ultrashort laser pulses are not completely understood. Here, we characterized photophysically this interaction on Hb thin film and erythrocytes using fluorescence spectroscopy upon single-photon/two-photon absorption, and UV-VIS single-photon absorption spectroscopy. A gradual increase of the fluorescence intensity, ending up with saturation, is observed upon prolonged exposure of Hb thin layer and erythrocytes to ultrashort laser pulses at 730 nm. When compared to protoporphyrin IX (PpIX) and oxidized Hb by H2O2, TPEF spectra from a thin Hb film and erythrocytes showed good mutual agreement, broad peaking at 550 nm, supporting hemoglobin undergoes degradation and that same fluorescent specie(s) originating from the heme moiety are generated. The uniform square shaped patterns of the fluorescent photoproduct exhibited the same level of the fluorescence intensity even after 12 weeks from the formation, indicating high photoproduct stability. We finally demonstrated the full potential of the formed Hb photoproduct with TPEF scanning microscopy towards spatiotemporally controlled micropatterning in HTF and single human erythrocyte labelling and tracking in the whole blood.

Label-free third harmonic generation imaging and quantification of lipid droplets in live filamentous fungi.

We report the utilization of Third-Harmonic Generation microscopy for label-free live cell imaging of lipid droplets in the hypha of filamentous fungus Phycomyces blakesleeanus. THG microscopy images showed bright spherical features dispersed throughout the hypha cytoplasm in control conditions and a transient increase in the number of bright features after complete nitrogen starvation. Colocalization analysis of THG and lipid-counterstained images disclosed that the cytoplasmic particles were lipid droplets. Particle Size Analysis and Image Correlation Spectroscopy were used to quantify the number density and size of lipid droplets. The two analysis methods both revealed an increase from 16 × 10-3 to 23 × 10-3 lipid droplets/µm2 after nitrogen starvation and a decrease in the average size of the droplets (range: 0.5-0.8 µm diameter). In conclusion, THG imaging, followed by PSA and ICS, can be reliably used for filamentous fungi for the in vivo quantification of lipid droplets without the need for labeling and/or fixation. In addition, it has been demonstrated that ICS is suitable for THG microscopy.

Polarization-resolved SHG imaging as a fast screening method for collagen alterations during aging: Comparison with light and electron microscopy.

Our previous study on rat skin showed that cumulative oxidative pressure induces profound structural and ultrastructural alterations in both rat skin epidermis and dermis during aging. Here, we aimed to investigate the biophotonic properties of collagen as a main dermal component in the function of chronological aging. We used second harmonic generation (SHG) and two-photon excited fluorescence (TPEF) on 5 µm thick skin paraffin sections from 15-day-, 1-month- and 21-month-old rats, respectively, to analyze collagen alterations, in comparison to conventional light and electron microscopy methods. Obtained results show that polarization-resolved SHG (PSHG) images can detect collagen fiber alterations in line with chronological aging and that this method is consistent with light and electron microscopy. Moreover, the ß coefficient calculated from PSHG images points out that delicate alterations lead to a more ordered structure of collagen molecules due to oxidative damage. The results of this study also open the possibility of successfully applying this fast and label-free method to previously fixed samples.

Altered organization of collagen fibers in the uninvolved human colon mucosa 10 cm and 20 cm away from the malignant tumor.

Remodelling of collagen fibers has been described during every phase of cancer genesis and progression. Changes in morphology and organization of collagen fibers contribute to the formation of microenvironment that favors cancer progression and development of metastasis. However, there are only few data about remodelling of collagen fibers in healthy looking mucosa distant from the cancer. Using SHG imaging, electron microscopy and specialized softwares (CT-FIRE, CurveAlign and FiberFit), we objectively visualized and quantified changes in morphology and organization of collagen fibers and investigated possible causes of collagen remodelling (change in syntheses, degradation and collagen cross-linking) in the colon mucosa 10 cm and 20 cm away from the cancer in comparison with healthy mucosa. We showed that in the lamina propria this far from the colon cancer, there were changes in collagen architecture (width, straightness, alignment of collagen fibers and collagen molecules inside fibers), increased representation of myofibroblasts and increase expression of collagen-remodelling enzymes (LOX and MMP2). Thus, the changes in organization of collagen fibers, which were already described in the cancer microenvironment, also exist in the mucosa far from the cancer, but smaller in magnitude.

Micromechanical imaging of dentin with Brillouin microscopy.

The structure of teeth can be altered by diet, age or diseases such as caries and sclerosis. It is very important to characterize their mechanical properties to predict and understand tooth decay, design restorative dental procedures, and investigate their tribological behavior. However, existing imaging techniques are not well suited to investigating the micromechanics of teeth, in particular at tissue interfaces. Here, we describe a microscope based on Brillouin light scattering (BLS) developed to probe the spectrum of the light scattered from tooth tissues, from which the mechanical properties (sound velocity, viscosity) can be inferred with a priori knowledge of the refractive index. BLS is an inelastic process that uses the scattering of light by acoustic waves in the GHz range. Our microscope thus reveals the mechanical properties at the micrometer scale without contact with the sample. BLS signals show significant differences between sound tissues and pathological lesions, and can be used to precisely delineate carious dentin. We also show maps of the sagittal and transversal planes of sound tubular dentin that reveal its anisotropic microstructure at 1 µm resolution. Our observations indicate that the collagen-based matrix of dentine is the main load-bearing structure, which can be considered as a fiber-reinforced composite. In the vicinity of polymeric tooth-filling materials, we observed the infiltration of the adhesive complex into the opened tubules of sound dentine. The ability to probe the quality of this interfacial layer could lead to innovative designs of biomaterials used for dental restorations in contemporary adhesive dentistry, with possible direct repercussions on decision-making during clinical work. STATEMENT OF SIGNIFICANCE: Mechanical properties of teeth can be altered by diet, age or diseases. Yet existing imaging modalities cannot reveal the micromechanics of the tooth. Here we developed a new type of microscope that uses the scattering of a laser light by naturally-occurring acoustic waves to probe mechanical changes in tooth tissues at a sub-micrometer scale without contact to the sample. We observe significant mechanical differences between healthy tissues and pathological lesions. The contrast in mechanical properties also reveals the microstructure of the polymer-dentin interfaces. We believe that this new development of laser spectroscopy is very important because it should lead to innovative designs of biomaterials used for dental restoration, and allow delineating precisely destructed dentin for minimally-invasive strategies.

Local redox conditions in cells imaged via non-fluorescent transient states of NAD(P)H.

The autofluorescent coenzyme nicotinamide adenine dinucleotide (NADH) and its phosphorylated form (NADPH) are major determinants of cellular redox balance. Both their fluorescence intensities and lifetimes are extensively used as label-free readouts in cellular metabolic imaging studies. Here, we introduce fluorescence blinking of NAD(P)H, as an additional, orthogonal readout in such studies. Blinking of fluorophores and their underlying dark state transitions are specifically sensitive to redox conditions and oxygenation, parameters of particular relevance in cellular metabolic studies. We show that such dark state transitions in NAD(P)H can be quantified via the average fluorescence intensity recorded upon modulated one-photon excitation, so-called transient state (TRAST) monitoring. Thereby, transitions in NAD(P)H, previously only accessible from elaborate spectroscopic cuvette measurements, can be imaged at subcellular resolution in live cells. We then demonstrate that these transitions can be imaged with a standard laser-scanning confocal microscope and two-photon excitation, in parallel with regular fluorescence lifetime imaging (FLIM). TRAST imaging of NAD(P)H was found to provide additional, orthogonal information to FLIM and allows altered oxidative environments in cells treated with a mitochondrial un-coupler or cyanide to be clearly distinguished. We propose TRAST imaging as a straightforward and widely applicable modality, extending the range of information obtainable from cellular metabolic imaging of NAD(P)H fluorescence.

Naturally safe: Cellular noise for document security.

Modern document protection relies on the simultaneous combination of many optical features with micron and submicron structures, whose complexity is the main obstacle for unauthorized copying. In that sense, documents are best protected by the diffractive optical elements generated lithographically and mass-produced by embossing. The problem is that the resulting security elements are identical, facilitating mass-production of both original and counterfeited documents. Here, we prove that each butterfly wing-scale is structurally and optically unique and can be used as an inimitable optical memory tag and applied for document security. Wing-scales, exhibiting angular variability of their color, were laser-cut and bleached to imprint cryptographic information of an authorized issuer. The resulting optical memory tag is extremely durable, as verified by several century-old insect specimens still retaining their coloration. The described technique is simple, amenable to mass-production, low cost and easy to integrate within the existing security infrastructure.

Photon diagnostics at the FLASH THz beamline.

The THz beamline at FLASH, DESY, provides both tunable (1-300 THz) narrow-bandwidth (∼10%) and broad-bandwidth intense (up to 150 uJ) THz pulses delivered in 1 MHz bursts and naturally synchronized with free-electron laser X-ray pulses. Combination of these pulses, along with the auxiliary NIR and VIS ultrashort lasers, supports a plethora of dynamic investigations in physics, material science and biology. The unique features of the FLASH THz pulses and the accelerator source, however, bring along a set of challenges in the diagnostics of their key parameters: pulse energy, spectral, temporal and spatial profiles. Here, these challenges are discussed and the pulse diagnostic tools developed at FLASH are presented. In particular, a radiometric power measurement is presented that enables the derivation of the average pulse energy within a pulse burst across the spectral range, jitter-corrected electro-optical sampling for the full spectro-temporal pulse characterization, spatial beam profiling along the beam transport line and at the sample, and a lamellar grating based Fourier transform infrared spectrometer for the on-line assessment of the average THz pulse spectra. Corresponding measurement results provide a comprehensive insight into the THz beamline capabilities.

NIR photo-driven upconversion in NaYF4:Yb,Er/PLGA particles for in vitro bioimaging of cancer cells.

Lanthanide-doped fluoride up-converting nanoparticles (UCNPs) represent the new class of imaging contrast agents which hold great potential for overcoming existing problems associated with traditionally used dyes, proteins and quantum dots. In this study, a new kind of hybrid NaYF4:Yb,Er/PLGA nanoparticles for efficient biolabeling were prepared through one-pot solvothermal synthesis route. Morphological and structural characteristics of the as-designed particles were obtained using X-ray powder diffraction (XRPD), scanning and transmission electron microscopy (SEM/TEM), energy dispersive spectroscopy (EDS), Fourier transform infrared (FTIR) and photoluminescence (PL) spectroscopy, while their cytotoxicity as well as up-conversion (UC) labeling capability were tested in vitro toward human gingival cells (HGC) and oral squamous cell carcinoma (OSCC). The results revealed coexistence of the cubic (Fm-3m) and hexagonal (P63/m) phase in spherical and irregularly shaped nanoparticles, respectively. PLGA [Poly(lactic-co-glycolic acid)] ligands attached at the surface of UCNPs particles provide their enhanced cellular uptake and enable high-quality cells imaging through a near-infrared (NIR) laser scanning microscopy (λex = 980 nm). Moreover, the fact that NaYF4:Yb,Er/PLGA UCNPs show low cytotoxicity against HGC over the whole concentration range (10-50 µg/mL) while a dose dependent viability of OSCC is obtained indicates that these might be a promising candidates for targeted cancer cell therapy.

One-step synthesis of amino-functionalized up-converting NaYF4:Yb,Er nanoparticles for in vitro cell imaging.

The emerging up-conversion nanoparticles (UCNPs) offer a wide range of biotechnology applications, from biomarkers and deep tissue imaging, to single molecule tracking and drug delivery. Their successful conjugation to biocompatible agents is crucial for specific molecules recognition and usually requires multiple steps which may lead to low reproducibility. Here, we report a simple and rapid one-step procedure for in situ synthesis of biocompatible amino-functionalized NaYF4:Yb,Er UCNPs that could be used for NIR-driven fluorescence cell labeling. X-ray diffraction showed that UCNPs synthesized through chitosan-assisted solvothermal processing are monophasic and crystallize in a cubic α phase. Scanning and transmission electron microscopy revealed that the obtained crystals are spherical in shape with a mean diameter of 120 nm. Photoluminescence spectra indicated weaker green (2H11/2, 4S3/2 → 4I15/2) and stronger red emission (4F9/2 → 4I15/2), as a result of enhanced non-radiative 4I11/2 → 4I13/2 Er3+ relaxation. The presence of chitosan groups at the surface of UCNPs was confirmed by Fourier transform infrared spectroscopy, thermogravimetry and X-ray photoelectron spectroscopy. This provides their enhanced internalization in cells, at low concentration of 10 µg ml-1, without suppression of cell viability after 24 h of exposure. Furthermore, upon 980 nm laser irradiation, the amino-functionalized NaYF4:Yb,Er UCNPs were successfully used in vitro for labeling of two human cell types, normal gingival and oral squamous cell carcinoma.

Remodeling of extracellular matrix of the lamina propria in the uninvolved human rectal mucosa 10 and 20 cm away from the malignant tumor.

In recent years, it has been demonstrated that malignancy arises and advances through the molecular interplay between tumor cells and non-malignant elements of the tumor stroma, that is, fibroblasts and extracellular matrix. However, in contrast to the mounting evidence about the role of tumor stroma in the genesis and progression of the malignant disease, there are very few data regarding the uninvolved stromal tissue in the remote surrounding of the tumor. Using the objective morphometric approach in patients with adenocarcinoma, we demonstrate the remodeling of extracellular matrix of the lamina propria in the uninvolved rectal mucosa 10 and 20 cm away from the neoplasm. We show that the representation of basic extracellular matrix constituents (reticular and collagen fibers and ground substance) is decreased. Also, the diameter of empty spaces that appear within the extracellular matrix of the lamina propria is increased. These spaces do not represent the blood or lymphatic vessel elements. Very likely, they reflect the development of tissue edema in the remote, uninvolved lamina propria of the mucosa in patients with the malignant tumor of the rectum. We hypothesize that the remodeling of extracellular matrix in lamina propria of the rectal mucosa may increase its stiffness, modulating the mechano-signal transduction, and thus promote the progression of the malignant disease.

Scattering-enhanced absorption and interference produce a golden wing color of the burnished brass moth, Diachrysia chrysitis.

Here we report how interference and scattering-enhanced absorption act together to produce the golden wing patches of the burnished brass moth. The key mechanism is scattering on rough internal surfaces of the wing scales, accompanied by a large increase of absorption in the UV-blue spectral range. Unscattered light interferes and efficiently reflects from the multilayer composed of the scales and the wing membranes. The resulting spectrum is remarkably similar to the spectrum of metallic gold. Subwavelength morphology and spectral and absorptive properties of the wings are described. Theories of subwavelength surface scattering and local intensity enhancement are used to quantitatively explain the observed reflectance spectrum.

Mapping of hemoglobin in erythrocytes and erythrocyte ghosts using two photon excitation fluorescence microscopy.

The present study describes utilization of two photon excitation fluorescence (2PE) microscopy for visualization of the hemoglobin in human and porcine erythrocytes and their empty membranes (i.e., ghosts). High-quality, label- and fixation-free visualization of hemoglobin was achieved at excitation wavelength 730 nm by detecting visible autofluorescence. Localization in the suspension and spatial distribution (i.e., mapping) of residual hemoglobin in erythrocyte ghosts has been resolved by 2PE. Prior to the 2PE mapping, the presence of residual hemoglobin in the bulk suspension of erythrocyte ghosts was confirmed by cyanmethemoglobin assay. 2PE analysis revealed that the distribution of hemoglobin in intact erythrocytes follows the cells' shape. Two types of erythrocytes, human and porcine, characterized with discocyte and echinocyte morphology, respectively, showed significant differences in hemoglobin distribution. The 2PE images have revealed that despite an extensive washing out procedure after gradual hypotonic hemolysis, a certain amount of hemoglobin localized on the intracellular side always remains bound to the membrane and cannot be eliminated. The obtained results open the possibility to use 2PE microscopy to examine hemoglobin distribution in erythrocytes and estimate the purity level of erythrocyte ghosts in biotechnological processes.

Nonlinear microscopy of chitin and chitinous structures: a case study of two cave-dwelling insects.

We performed a study of the nonlinear optical properties of chemically purified chitin and insect cuticle using two-photon excited autofluorescence (TPEF) and second-harmonic generation (SHG) microscopy. Excitation spectrum, fluorescence time, polarization sensitivity, and bleaching speed were measured. We have found that the maximum autofluorescence signal requires an excitation wavelength below 850 nm. At longer wavelengths, we were able to penetrate more than 150-um deep into the sample through the chitinous structures. The excitation power was kept below 10 mW (at the sample) in order to diminish bleaching. The SHG from the purified chitin was confirmed by spectral- and time-resolved measurements. Two cave-dwelling, depigmented, insect species were analyzed and three-dimensional images of the cuticular structures were obtained.