Minimizing photodecomposition of flavin adenine dinucleotide fluorescence by the use of pulsed LEDs

Dynamic alterations in flavin adenine dinucleotide (FAD) fluorescence permit insight into energy metabolism‐dependent changes of intramitochondrial redox potential. Monitoring FAD fluorescence in living tissue is impeded by photobleaching, restricting the length of microfluorimetric recordings. In addition, photodecomposition of these essential electron carriers negatively interferes with energy metabolism and viability of the biological specimen. Taking advantage of pulsed LED illumination, here we determined the optimal excitation settings giving the largest fluorescence yield with the lowest photobleaching and interference with metabolism in hippocampal brain slices. The effects of FAD bleaching on energy metabolism and viability were studied by monitoring tissue pO₂, field potentials and changes in extracellular potassium concentration ([K⁺]_o). Photobleaching with continuous illumination consisted of an initial exponential decrease followed by a nearly linear decay. The exponential decay was significantly decelerated with pulsed illumination. Pulse length of 5 ms was sufficient to reach a fluorescence output comparable to continuous illumination, whereas further increasing duration increased photobleaching. Similarly, photobleaching increased with shortening of the interpulse interval. Photobleaching was partially reversible indicating the existence of a transient nonfluorescent flavin derivative. Pulsed illumination decreased FAD photodecomposition, improved slice viability and reproducibility of stimulus‐induced FAD, field potential, [K⁺]_o and pO₂ changes as compared to continuous illumination.     

Reconstructing the microstructure of polyimide–silicalite mixed‐matrix membranes and their particle connectivity using FIB‐SEM tomography

Properties of a composite material made of a continuous matrix and particles often depend on microscopic details, such as contacts between particles. Focusing on processing raw focused‐ion beam scanning electron microscope (FIB‐SEM) tomography data, we reconstructed three mixed‐matrix membrane samples made of 6FDA‐ODA polyimide and silicalite‐1 particles. In the first step of image processing, backscattered electron (BSE) and secondary electron (SE) signals were mixed in a ratio that was expected to obtain a segmented 3D image with a realistic volume fraction of silicalite‐1. Second, after spatial alignment of the stacked FIB‐SEM data, the 3D image was smoothed using adaptive median and anisotropic nonlinear diffusion filters. Third, the image was segmented using the power watershed method coupled with a seeding algorithm based on geodesic reconstruction from the markers. If the resulting volume fraction did not match the target value quantified by chemical analysis of the sample, the BSE and SE signals were mixed in another ratio and the procedure was repeated until the target volume fraction was achieved. Otherwise, the segmented 3D image (replica) was accepted and its microstructure was thoroughly characterized with special attention paid to connectivity of the silicalite phase. In terms of the phase connectivity, Monte Carlo simulations based on the pure‐phase permeability values enabled us to calculate the effective permeability tensor, the main diagonal elements of which were compared with the experimental permeability. In line with the hypothesis proposed in our recent paper (?apek, P. et al. (2014) Comput. Mater. Sci. 89 , 142–156), the results confirmed that the existence of particle clusters was a key microstructural feature determining effective permeability.     

Sequential processing of quantitative phase images for the study of cell behaviour in real‐time digital holographic microscopy

Transmitted light holographic microscopy is particularly used for quantitative phase imaging of transparent microscopic objects such as living cells. The study of the cell is based on extraction of the dynamic data on cell behaviour from the time‐lapse sequence of the phase images. However, the phase images are affected by the phase aberrations that make the analysis particularly difficult. This is because the phase deformation is prone to change during long‐term experiments. Here, we present a novel algorithm for sequential processing of living cells phase images in a time‐lapse sequence. The algorithm compensates for the deformation of a phase image using weighted least‐squares surface fitting. Moreover, it identifies and segments the individual cells in the phase image. All these procedures are performed automatically and applied immediately after obtaining every single phase image. This property of the algorithm is important for real‐time cell quantitative phase imaging and instantaneous control of the course of the experiment by playback of the recorded sequence up to actual time. Such operator's intervention is a forerunner of process automation derived from image analysis. The efficiency of the propounded algorithm is demonstrated on images of rat fibrosarcoma cells using an off‐axis holographic microscope.     

Mechanical Properties of Pumpkin

Mechanical properties of three common varieties of pumpkin were evaluated and statistically compared. Toughness, rupture force, shear strength, and cutting force were determined for Jarrahdale, Jap, and Butternut varieties. The investigation was carried out in three cases of flesh, skin and unpeeled product, ignoring the toughness and rupture force of flesh. Relative contribution of skin to unpeeled case of each property was estimated. Varieties were found statistically alike in rupture force, toughness, and maximum shear strength force of unpeeled cases. Also the skin of three varieties showed similar strength in shear (p>0.05). Jap and Butternut varieties for some other properties showed close values. Maximum shear strength force of flesh, shear strength of unpeeled case, and relative contribution of skin to shear strength of unpeeled case were close (p>0.05) for these varieties. Jarrahdale had no difference in shear strength of flesh with two other varieties. It was also similar (p>0.05) to Jap in relative contribution of skin to shear strength, rupture force, and toughness of unpeeled case.     

Characterization of composite materials based on Fe powder (core) and phenol–formaldehyde resin (shell) modified with nanometer-sized SiO2

Soft magnetic composites based on Fe powder and phenol–formaldehyde resin (PFR) modified with tetraethylorthosilicate are investigated in detail. The chemical synthesis of PFR, its modification with nanometer-sized SiO₂ particles created by sol–gel method and subsequent coating, enables a preparation of insulating PFR–SiO₂ (PFRT) layer on the surface of Fe particles. Thermal degradation and FTIR analysis of PFR and PFRT with different amount of SiO₂ was examined. Mechanical hardness and flexural strength of FePFRT composites was studied depending on the amount of nanosized-SiO₂ in the coating. SEM serves in evidence of a defectless microstructure if the coating contains at least 2% of silica particles. The morphology of Fe particles implies uniform coating without any visible exfoliation. A presence of fine SiO₂ particles was verified by TEM. The best magnetic properties were found in Fe–PFRT composite with 2% of SiO₂ in the insulating layer on behalf of its uniform arrangement and homogeneity.     

Adhesion and proliferation of keratinocytes on ion beam modified polyethylene

Polyethylene (PE) foils were modified by irradiation with ArE ⁺ and XeE ⁺ ions to different fluences and different physico-chemical properties of the irradiated PE were studied in relation to adhesion and proliferation of keratinocytes on the modified surface. Changes in the PE surface roughness were examined using the AFM technique, the production of conjugated double bonds and oxidized structures by UV-VIS and FTIR techniques respectively. The surface polarity was determined by measuring surface contact angle and two-point technique was used for the determination of PE sheet resistance. Adhesion and proliferation of keratinocytes was characterized using the MTT-test. The ion irradiation leads to creation of conjugated double bonds which, together with progressive carbonization, contribute to the observed decrease of sheet resistance. Oxidation of the irradiated PE surface layer during the ion implantation is observed. Besides oxidation, the PE surface polarity is affected by other factors. The observed increase of the PE surface roughness due to the ion irradiation is inversely proportional to the ion size. The adhesion and proliferation of keratinocytes on the ion irradiated PE is significantly higher than on the pristine PE. Distribution of results in keratinocyte cultivation and the number of cells is related to the ion fluence applied and to ion species as well.     

Advantages and pitfalls of using free‐hand sections of frozen needles for three‐dimensional analysis of mesophyll by stereology and confocal microscopy

The anatomical structure of mesophyll tissue in the leaf is tightly connected with many physiological processes in plants. One of the most important mesophyll parameters related to photosynthesis is the internal leaf surface area, i.e. the surface area of mesophyll cell walls exposed to intercellular spaces. An efficient design‐based stereological method can be applied for estimation of this parameter, using software‐randomized virtual fakir test probes in stacks of optical sections acquired by a confocal microscope within thick physical free‐hand sections (i.e. acquired using a hand microtome), as we have shown in the case of fresh Norway spruce needles recently. However, for wider practical use in plant ecophysiology, a suitable form of sample storage and other possible technical constraints of this methodology need to be checked. We tested the effect of freezing conifer needles on their anatomical structure as well as the effect of possible deformations due to the cutting of unembedded material by a hand microtome, which can result in distortions of cutting surfaces. In the present study we found a higher proportion of intercellular spaces in mesophyll in regions near to the surface of a physical section, which means that the measurements should be restricted only to the middle region of the optical section series. On the other hand, the proportion of intercellular spaces in mesophyll as well as the internal needle surface density in mesophyll did not show significant difference between fresh and frozen needles; therefore, we conclude that freezing represents a suitable form of storage of sampled material for proposed stereological evaluation.     

Optimization of a quantitative method for muscle histology assessment

Optical microscopy offers the simplest way to obtain magnified images of biological tissues. The assessment of the muscle destructuration level can be performed by a method called Meat Destruction Indicator (MDI), which combines optical microscopy and image analysis. MDI can be used for evaluation of food quality and for considering mechanically separated meat (meat raw material with an MDI value above 58.1% contained muscle fibres sufficiently destructured). This paper is particularly focused on the metrological optimization of a quantitative image analysis method around the example of MDI measurement by microscopy, especially on the digital acquisition calibration focusing and analysis work‐flow. Ten different samples (45 sections) were examined with variable settings of microscope and camera to define the optimal configuration. The tests were performed with different observers to define rules and criteria for results validation. Based on the obtained results, we suggest choosing objective rules to set the light and colour of the camera and the microscope focus. To control the results of the automatic segmentation emerged also as a key step, and objective rules for observers to select or discard wrong segmented images should be defined. The adjusted MDI measurement by microscope can be used as a reliable method with good repeatability, thanks to this metrological assessment, which could and should be applied to all image analysis applications whatever the application.