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.
We describe a method for dual-view biomechanical strain measurements of highly asymmetrical biological objects, like teeth or bones. By using a spherical mirror, we were able to simultaneously record ...a digital hologram of the object itself and the mirror image of its (otherwise invisible) rear side. A single laser beam was sufficient to illuminate both sides of the object, and to provide a reference beam. As a result, the system was mechanically very stable, enabling long exposure times (up to 2 min) without the need for vibration isolation. The setup is simple to construct and adjust, and can be used to interferometrically observe any object that is smaller than the mirror diameter. Parallel data processing on a CUDA-enabled (compute unified device architecture) graphics card was used to reconstruct digital holograms and to further correct image distortion. We used the setup to measure the deformation of a tooth due to mastication forces. The finite-element method was used to compare experimental results and theoretical predictions.
Microoptical components are coming of age in a wide range of applications: lab-on-a-chip, imaging, detection… There are a large number of fabrication technologies capable of producing high quality ...individual components and their arrays. However, most of them require high-end and costly equipment, complex and time-consuming fabrication, harmful chemicals, resulting in expensive final products. Here we present a technology capable of producing high quality microoptical components, using low-end direct laser writing on a biocompatible, environmentally friendly hydrogel, without any waste substances. The gel is locally and controllably melted while surface tension forces shape the optical component, following the laser beam profile. The process is so quick that a single microlens is fabricated in less than a second, and can be used instantly without any further processing. The technology is neither subtractive nor additive, and the base material is simply displaced producing a smooth surface. We have been able to fabricate individual microlenses and their arrays (positive, negative, aspheric), gratings and diffractive components. The technology is tested by generating unique, difficult to counterfeit QR-codes. Turnaround time is fast and makes the technology suitable both for rapid prototyping and serial production.
The influence of material characteristics—i.e., type or surface texture—to wetting properties is nowadays increased by the implementation of ultrafast lasers for nanostructuring. In this account, we ...exposed multilayer thin metal film samples of different materials to a femtosecond laser beam at a 1030 nm wavelength. The interaction generated high-quality laser-induced periodic surface structures (LIPSS) of spatial periods between 740 and 790 nm and with maximal average corrugation height below 100 nm. The contact angle (CA) values of the water droplets on the surface were estimated and the values between unmodified and modified samples were compared. Even though the laser interaction changed both the surface morphology and the chemical composition, the wetting properties were predominantly influenced by the small change in morphology causing the increase in the contact angle of ~80%, which could not be explained classically. The influence of both surface corrugation and chemical composition to the wetting properties has been thoroughly investigated, discussed and explained. The presented results clearly confirm that femtosecond patterning can be used to mold wetting properties.
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.
Three subterranean leptodirine leiodid taxa, viz., Bozidaria Ćurčić & Pavićević gen. nov., Bozidaria serbooccidentalis Ćurčić & Pavićević gen. et sp. nov. and Proleonhardella (Proleonhardella) ...tarensis Ćurčić & Pavićević sp. nov., are described and diagnosed. Bozidaria Ćurčić & Pavićević gen. nov. belongs to the phyletic series of “Leonhardella”. The new beetle taxa differ from their closest relatives in numerous morphological characters. They most likely belong to phyletic lineages of Pliocene age. The new leiodid taxa are endemic to the Dinaric mountain chain of western Serbia. Keys to the leptodirine leiodid genera of the phyletic series of “Leonhardella” and to the taxa of the genus Proleonhardella Jeannel, 1910 are included.
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.
•SVD-COPULA is a method for denoising very low count two-photon laser-scanned microscopic images.•It reduces image acquisition time by reducing the stack of repeated microscopic images.•It is ...completely automatic, based on singular value decomposition with a two-stage copula threshold.•Its performance is stable in any image stack with mixed Poisson-Gaussian noise.
Multi-photon laser scanning microscopy is an advantageous technique for layered imaging in thick tissue samples. It enables reduced phototoxicity as the region of fluorescence excitation is limited. But, the small number and stochastic nature of detected photons require the multifold acquisition of each image so the photodamage may accumulate. This paper aims to reduce the number of repeated image acquisitions, proposing a denoising technique based on singular value decomposition with a novel two-stage copula threshold, thus the name SVD-COPULA. The first stage defines the referent noise level based on copula analysis. Following the comparative analysis of classical techniques and different copula families, Frank's copula is selected to determine the regions for noise estimation. The second threshold stage analyzes the stationarity of the singular values and adjusts the number of the eigenimages necessary for reconstruction, an issue important for images without the distinctive background regions. A problem of the mixed discrete-continual nature of pixel values is noticed and resolved. The procedure is fully automatic, with the built-in set of parameters and without the necessity for prior image analysis. The proposed method, together with twelve state-of-the-art denoising methods, are tested using the microscopic images from the proprietary and open databases, and the test images with simulated mixed Poisson-Gaussian noise. It is shown that the performance of SVD-COPULA is consistently good for all investigated images.
The iridescent features of the butterfly species Apatura iris (Linnaeus, 1758) and A. ilia (Denis & Schiffermüller, 1775) were studied. We recognized the structural color of scales only on the dorsal ...side of both the fore and hind wings of males of both of the aforementioned butterfly species. The scale dimensions and microstructure were analyzed by a scanning electron microscope (SEM) and transmission electron microscope (TEM). The optical properties were measured and it was found that the peak reflectivity is around 380 nm, with a spectral width (full width at half maximum) of approximately 50 nm in both species. The angular selectivity is high and a purple iridescent color is observed within the angular range of only 18 degrees in both species.
There is an abundance of plastic materials used in the widest range of applications, such as packaging, machine parts, biomedical devices and components, etc. However, most materials used today are ...non-decomposable in the environment, producing a huge burden on ecosystems. The search for better, safer alternatives is still on. Here we present a detailed analysis of a simple, cheap, non-toxic, even edible, eco-friendly material, which can be easily manufactured, laser patterned and used for the fabrication of complex structures. The base substance is gelatin which is made photoresponsive by adding plasticizers and sensitizers. The resulting films were analyzed with respect to their optical, thermal and mechanical properties, which can be modified by a slight variation of chemical composition. The material is optimized for rapid laser-manufacturing of elastic microstructures (lenses, gratings, cantilevers, etc.) without any waste or residues. Overall, the material properties were tailored to increase photothermal responsivity, improve the surface quality and achieve material homogeneity, transparency and long-term stability (as verified using electron microscopy, infrared spectroscopy and differential scanning calorimetry).
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