Rapid and label‐free detection and assessment of bacteria by terahertz time‐domain spectroscopy

Here we demonstrated the potential and applicability of terahertz (THz) spectroscopy to detect four commonly found bacteria in the infectious diseases. Besides the different spectral characteristics between bacterial species, THz absorption differences for living bacteria, dead bacteria and bacterial powder of the same species were also investigated. Our results revealed that small differences in water contents between bacterial cells account for distinct discrepancies of the absorption coefficients, which can be used for bacterial species identification. Furthermore, living and dead bacteria showed different absorption coefficients as a result of their different hydration levels, suggesting that THz spectroscopy can be used to rapidly assess the living state of bacteria under test. Our results clearly demonstrated the ability of THz spectroscopy for time‐saving and label‐free detection of bacteria with minimal sample preparation, potentially to be utilized for point‐of‐care tests in the near future.

Schematic representation of bacterial detection by THz spectroscopy. Different bacteria have distinctive absorption coefficients as a result of their different water contents.     

Label‐free analysis of mononuclear human blood cells in microfluidic flow by coherent imaging tools

The investigation of the physical properties of peripheral blood mononuclear cells (PBMC) is of great relevance, as they play a key role in regulating human body health. Here we report the possibility to characterize human PBMC in their physiological conditions in a microfluidic‐based measurement system. A viscoelastic polymer solution is adopted for 3D alignment of individual cells inflow. An optical signature (OS) acquisition of each flowing cell is performed using a wide angle light scattering apparatus. Besides, a quantitative phase imaging (QPI) holographic system is employed with the aim (i) to check the position in flow of individual cells using a holographic 3D cell tracking method; and (ii) to estimate their 3D morphometric features, such as their refractive index (RI). Results obtained by combining OS and QPI have been compared with literature values, showing good agreement. The results confirm the possibility to obtain sub‐micrometric details of physical cell properties in microfluidic flow, avoiding chemical staining or fluorescent labelling.

     

DeepTetrad: high‐throughput image analysis of meiotic tetrads by deep learning in Arabidopsis thaliana

Meiotic crossovers facilitate chromosome segregation and create new combinations of alleles in gametes. Crossover frequency varies along chromosomes and crossover interference limits the coincidence of closely spaced crossovers. Crossovers can be measured by observing the inheritance of linked transgenes expressing different colors of fluorescent protein in Arabidopsis pollen tetrads. Here we establish DeepTetrad, a deep learning‐based image recognition package for pollen tetrad analysis that enables high‐throughput measurements of crossover frequency and interference in individual plants. DeepTetrad will accelerate the genetic dissection of mechanisms that control meiotic recombination.     

Label‐free and non‐invasive discrimination of HaCaT and melanoma cells in a co‐culture model by hyperspectral confocal reflectance microscopy

A novel hyperspectral confocal microscopy method to separate different cell populations in a co‐culture model is presented here. The described methodological and instrumental approach allows discrimination of different cell types using a non‐invasive, label free method with good accuracy with a single cell resolution. In particular, melanoma cells are discriminated from HaCaT cells by hyperspectral confocal imaging, principal component analysis and optical frequencies signing, as confirmed by fluorescence labelling cross check. The identification seems to be quite robust to be insensitive to the cellular shape within the studied samples, enabling to separate cells according to their cytotype down to a single cell sensitivity.

Set of hyperspectral images of melanoma‐keratinocytes co‐culture model (left), score plot of principal component analysis and spectral analysis of principal components coefficients (center), label‐free spectral identification of cell populations (right).     

Evolution of gold nanoparticle clusters in living cells studied by sectional dark‐field optical microscopy and chromatic analysis

The evolution of gold nanoparticle (Au NP) clusters in living cells are studied by using sectional dark‐field optical microscopy and chromatic analysis approach. During endocytosis, Au NP clusters undergo fantastic color changes, from green to yellow‐orange due to the plasmonic coupling effect. Analysis of brightness/hue values of the dark‐field images helps estimate the numbers of Au NPs in the clusters. The Au NP clusters were further categorized into four groups within the endocytosis. As the results, the late endosomes had increased number of large Au NP clusters with time, while clustered numbers in secondary and tertiary groups were first increased and then decreased due to the fusion and fission of the endocytic vesicles. The time constants and cluster numbers for different groups are fitted by using an integrated rate equation, and show a positive correlation with the size of the Au NP cluster. The efficiency of Au NP uptake is only about 50% for normal cells, while 75% for cancer cells. Compared to normal cells, cancer cells show a larger number in uptake, while faster rate in removal. The propose method helps the kinetic study of endocytosed nanoparticles in physiological conditions.

     

Transformer‐4 version 2.0.1, a free multi‐platform software to quickly reformat genotype matrices of any marker type,and archive them in the Demiurge information system

Transformer‐4 version 2.0.1 (T4) is a multi‐platform freeware programmed in java that can transform a genotype matrix in Excel or XML format into the input formats of one or several of the most commonly used population genetic software, for any possible combination of the populations that the matrix contains. T4 also allows the users to (i) draw allozyme gel interpretations for any number of diploid individuals, and then generate a genotype matrix ready to be used by T4; and (ii) produce basic reports about the data in the matrices. Furthermore, T4 is the only way to optionally submit ‘genetic diversity digests’ for publication in the Demiurge online information system ( http://www.demiurge-project.org ). Each such digest undergoes peer‐review, and it consists of a geo‐referenced data matrix in the tfm4 format plus any ancillary document or hyperlink that the digest authors see fit to include. The complementarity between T4 and Demiurge facilitates a free, safe, permanent, and standardized data archival and analysis system for researchers, and may also be a convenient resource for scientific journals, public administrations, or higher educators. T4 and its converters are freely available (at, respectively, http://www.demiurge-project.org/download_t4 and http://www.demiurge-project.org/converterstore ) upon registration in the Demiurge information system ( http://demiurge-project.org/register ). Users have to click on the link provided on an account validation email, and accept Demiurge's terms of use (see http://www.demiurge-project.org/termsofuse ). A thorough user's guide is available within T4. A 3‐min promotional video about T4 and Demiurge can be seen at http://vimeo.com/29828406 .     

Automatic and label‐free identification of blood vessels in gliomas using the combination of multiphoton microscopy and image analysis

Currently, the targeted treatment of tumor based on the tumor microenvironment is newly developed. Blood vessels are the key parts in the tumor microenvironment, which is taken as a new visible target for tumor therapy. Multiphoton microscopy (MPM), based on the second harmonic generation and two‐photon excited fluorescence, is available to make the label‐free analysis on the blood vessels in human gliomas. MPM can reveal the vascular morphological characteristics in gliomas, including vascular malformation, intense vascular proliferation, perivascular collagen deposition, perivascular lymphocytes aggregation and microvascular proliferation. In addition, the image analysis algorithms were developed to automatically calculate the perivascular collagen content, vascular cavity area, lumen area, wall area and vessel number. Thus, the vascular morphology, the perivascular collagen deposition and intense vascular proliferation degree can be further quantitatively characterized. Compared with the pathological analysis, the combination of MPM and image analysis has potential advantages in making a quantitative and qualitative analyzing on vascular morphology in glioma microenvironment. As micro‐endoscope and two‐photon fiberscope are technologically improved, this combined method will be a useful imaging way to make the real‐time research on the targeting tumor microenvironment in gliomas.     

Quantitative analysis of safranal in saffron extract and nanoparticle formulation by a validated high‐performance thin‐layer chromatographic method†

Introduction – Safranal is an effective anticonvulsant shown to act as an agonist at GABA_A receptors. Nose to brain delivery via nanoparticle formulation might improve its brain delivery. A selective and sensitive analytical method is required for evaluation of safranal‐based novel drug delivery systems. Objective – To develop and validate a high‐performance thin‐layer chromatographic (HPTLC) method for the quantitative analysis of safranal as bulk, in saffron extract and in developed safranal‐loaded nanoparticle formulation. Methodology – Chromatographic separation was achieved on silica gel pre‐coated TLC aluminium plates 60F‐254, using n‐hexane:ethyl acetate (9 : 1, v/v) as the mobile phase. Quantitative analysis was carried out by densitometry at a wavelength of 310 nm. The method was validated and applied to detect related impurities, to analyse safranal in saffron extract and to evaluate safranal‐loaded nanoparticles. Results – Compact spots of safranal were observed at R_f value 0.51 ± 0.02. The method was linear (r = 0.9991) between 0.5 and 5.0 μg/spot. The intra‐ and inter‐day precisions were 1.08–2.17 and 1. 86–3.47%, respectively. The limit of detection was 50 ng/spot and the limit of quantification was 150 ng/spot. The method proved to be accurate (recovery 97.4–102.0%) and was selective for safranal. Evaluation of safranal‐loaded nanoparticle formulation demonstrated drug loading of 23.0%, encapsulation efficiency of 42.0% and sustained drug release following biphasic pattern. Conclusion – The present method is useful for the quantitative and qualitative analysis of safranal and safranal‐loaded nanoparticle formulation. It provides significant advantages in terms of greater specificity and rapid analysis. Copyright © 2009 John Wiley & Sons, Ltd.     

Digitized photographs in vegetation analysis ‐ a comparison of cover estimates

Abstract. The use of digital images in measuring plant species cover and species number in boreal forest vegetation was studied. Plant cover was estimated by manual delineation on photographs and subsequent digitized measurement of the areas. This value was regarded as the reference and compared with cover obtained by visual estimate, point frequency and automatic image analysis methods. The automatic image analysis was based on scanned photographs. Supervised image classification with ERDAS software was then used to distinguish between the covers of different plant species. When comparing the ability of the methods to detect species, the visual estimate method gave values similar to the reference. The study material was collected from three sites along a heavy‐metal pollution transect in western Finland. All four methods detected, in a similar manner, differences between the plant species abundances along the transect. Compared with the reference, the digital images underestimated the cover of the lichens Cladina spp. and Cetraria islandica, but gave similar estimates for the dwarf shrubs Empetrum nigrum and Vaccinium vitis‐idaea. The point frequency method overestimated the cover of all the species studied. The visual estimates of lichens were close to the reference, while the dwarf shrub covers were overestimated. The number of species detected using supervised image analysis and the point frequency method was lower than that with visual estimation. Visual estimation was faster, and the estimate closer to the reference cover values than the others. Digital images may be useful in detecting changes in some selected species in vegetation with a simple vertical structure but with taller, multilayered vegetation and a higher species number, the reliability of the cover estimates is lower.     

Enantioselective analysis of ibuprofen enantiomers in mice plasma and tissues by high‐performance liquid chromatography with fluorescence detection: Application to a pharmacokinetic study

A direct fluorometric high‐performance liquid chromatography (HPLC) method was developed and validated for the analysis of ibuprofen enantiomers in mouse plasma (100 μl) and tissues (brain, liver, kidneys) using liquid–liquid extraction and 4‐tertbutylphenoxyacetic acid as an internal standard. Separation of enantiomers was accomplished in a Chiracel OJ‐H chiral column based on cellulose tris(4‐methylbenzoate) coated on 5 μm silica‐gel, 250 x 4.6 mm at 22 °C with a mobile phase composed of n‐hexane, 2‐propanol, and trifluoroacetic acid that were delivered in gradient elution at a flow rate of 1 ml min⁻¹. A fluorometric detector was set at: λ_excit. = 220 nm and λ_emis. = 290 nm. Method validation included the evaluation of the selectivity, linearity, lower limit of quantification (LLOQ), within‐run and between‐run precision and accuracy. The LLOQ for the two enantiomers was 0.125 μg ml⁻¹ in plasma, 0.09 μg g⁻¹ in brain, and 0.25 μg g⁻¹ in for liver and kidney homogenates. The calibration curves showed good linearity in the ranges of each enantiomers: from 0.125 to 35 μg ml⁻¹ for plasma, 0.09–1.44 μg g⁻¹ for brain, and 0.25–20 μg g⁻¹ for liver and kidney homogenates. The method was successfully applied to a pharmacokinetic study of ibuprofen enantiomers in mice treated i.v. with 10 mg kg⁻¹ of racemate.