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Total-internal reflection fluorescence (TIRF) microscope is a unique technique for selective excitation of only those fluorophore molecules in a cellular environment, which are located at the sub-diffraction axial distance of a cell's contact-area. Despite this prominent feature of the TIRF microscope, making quantitative use of this technique has been a challenge, since the excitation intensity strongly depends on the axial position of a fluorophore molecule. Here, we present an easy-implemented data analysis method to quantitatively characterize the fluorescent signal, without considering the intensity-value. We use F-actin patches in single-melanoma cells as an example and define two quantities of elongation and surface density for F-actin patches at the contact-area of a melanoma cell. The elongation parameter can evaluate the dispersion of F-actin patches at the contact-area of a cell and is useful to classify the attaching, spreading, and expanding stages of a cell. Following that, we present the profile of the surface density of F-actin patches as a quantity to probe the spatio-temporal distribution of the F-actin patches at the contact-area of a cell. The data analysis methods that are proposed here will also be applicable in the image analysis of the other advanced optical microscopic methods., (© 2022. The Author(s).)

A combination of light microscopy and image processing was applied to investigate morphology of label-free primary-melanocytes and melanoma cells. A novel methodological approach based on morphology of nuclear body was used to find those single cells, which were at the same phase of cell cycle. The area and perimeter of melanocytes and melanoma cells were quantified. We found that there was a significant difference between area and perimeter of adendritic-shaped melanocytes with melanoma cells and the reason(s) of this finding was speculated. Finally, a theoretical model based on losing dendrites was proposed, which was in agreement with our experimental data.

Laboratory assays such as MIC tests assume that antibiotic molecules are stable in the chosen growth medium-but rapid degradation has been observed for antibiotics including β-lactams under some conditions in aqueous solution. Degradation rates in bacterial growth medium are less well known. Here, we develop a 'delay time bioassay' that provides a simple way to estimate antibiotic stability in bacterial growth media, using only a plate reader and without the need to measure the antibiotic concentration directly. We use the bioassay to measure degradation half-lives of the β-lactam antibiotics mecillinam, aztreonam and cefotaxime in widely-used bacterial growth media based on MOPS and Luria-Bertani (LB) broth. We find that mecillinam degradation can occur rapidly, with a half-life as short as 2 hours in MOPS medium at 37°C and pH 7.4, and 4-5 hours in LB, but that adjusting the pH and temperature can increase its stability to a half-life around 6 hours without excessively perturbing growth. Aztreonam and cefotaxime were found to have half-lives longer than 6 hours in MOPS medium at 37°C and pH 7.4, but still shorter than the timescale of a typical minimum inhibitory concentration (MIC) assay. Taken together, our results suggest that care is needed in interpreting MIC tests and other laboratory growth assays for β-lactam antibiotics, since there may be significant degradation of the antibiotic during the assay., Competing Interests: The authors have declared that no competing interests exist.

In a total-internal-reflection-fluorescence-microscopy method, there is anisotropy in the polarized evanescent wave. Since the evanescent wave is used as an excitation field, the mentioned anisotropy is a disadvantage in using the total-internal-reflection-fluorescence-microscopy technique. Therefore, by theoretical and analytical approaches, and based on the Fresnel coefficients, the effect of three dielectrics media on the anisotropy of the evanescent wave is investigated. Following that, a proper combination of the cover glass, oil immersion, and prism for both living and non-living samples is suggested that not only enhances the intensity of the evanescent wave, but also and importantly, decreases the essential anisotropy of the evanescent wave.

Expansion of human induced pluripotent stem cells (h-iPSCs) on mouse derived feeder layers or murine cells secretions such as Matrigel hamper their clinical applications. Alternative methods have introduced novel substrates as stem cell niches or/and optimized combinations of humanized soluble factors as fully defined mediums. Accordingly vitronectin as a main part of ECM have been commercialized significantly as a stem cell niche-forming substrate. In this work, we used a functional peptide derived from vitronectin (VTN) and co-immobilized it with FGF-2 (as an indisputable ingredient of defined culture mediums) on chitosan film surface. After chemical and physical characterization of the pristine chitosan surface as well as ones modified by VTN or/and FGF-2, h-iPS cells were cultured on them at the xeno/feeder-free conditions. Our results demonstrated that co-immobilization of these two biomolecules has a synergistic effect on adhesion and clonal growth of h-iPS cells with maintained expression of pluripotency markers in a FGF-2 density-dependent manner. This is the first report of co-immobilization of an ECM derived molecule and a growth factor for stem cell culture., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A combination of light-microscopy and image processing was used to elaborate on the fluctuation in the width of the cylindrical part of Escherichia coli at sub-pixel-resolution, and under in vivo conditions. The mean-squared-width-difference along the axial direction of the cylindrical part of a number of bacteria was measured. The results reveal that the cylindrical part of Escherichia coli is composed of multi-domain morphological structures. The length of the domains starts at 150 nm in newborn cells, and linearly increases in length up to 300 nm in aged cells. The fluctuation in the local-cell-widths in each domain is less than the fluctuation of local-cell-widths between different domains. Local cell width correlations along the cell body occur on a length scale of less than 50 nm. This finding could be associated with the flexibility of the cell envelope in the radial versus longitudinal directions.

A combination of light-microscopy and image processing has been applied to study naturally deformed Escherichia coli under in vivo condition and at the order of sub-pixel high-resolution accuracy. To classify deflagellated non-dividing E. coli cells to the rod-shape and bent-shape, a geometrical approach has been applied. From the analysis of the geometrical data which were obtained of image processing, we estimated the required effective energy for shaping a rod-shape to a bent-shape with the same size. We evaluated the energy of deformation in the naturally deformed bacteria with minimum cell manipulation, under in vivo condition, and with minimum influence of any external force, torque and pressure. Finally, we have also elaborated on the possible scenario to explain how naturally deformed bacteria are formed from initial to final-stage.

A novel design of gold-coated iron oxide nanoparticles was fabricated as a potential delivery system to improve the efficiency and stability of d, l-sulforaphane as an anticancer drug. To this purpose, the surface of gold-coated iron oxide nanoparticles was modified for sulforaphane delivery via furnishing its surface with thiolated polyethylene glycol-folic acid and thiolated polyethylene glycol-FITC. The synthesized nanoparticles were characterized by different techniques such as FTIR, energy dispersive X-ray spectroscopy, UV-visible spectroscopy, scanning and transmission electron microscopy. The average diameters of the synthesized nanoparticles before and after sulforaphane loading were obtained ∼ 33 nm and ∼ 38 nm, respectively, when ∼ 2.8 mmol/g of sulforaphane was loaded. The result of cell viability assay which was confirmed by apoptosis assay on the human breast cancer cells (MCF-7 line) as a model of in vitro-cancerous cells, proved that the bare nanoparticles showed little inherent cytotoxicity, whereas the sulforaphane-loaded nanoparticles were cytotoxic. The expression rate of the anti-apoptotic genes (bcl-2 and bcl-xL), and the pro-apoptotic genes (bax and bak) were quantified, and it was found that the expression rate of bcl-2 and bcl-xL genes significantly were decreased when MCF-7 cells were incubated by sulforaphane-loaded nanoparticles. The sulforaphane-loaded into the designed gold-coated iron oxide nanoparticles, acceptably induced apoptosis in MCF-7 cells.