The processing of staphylococcal biofilm images from Fluorescence and Confocal Microscopy to assess in vitro efficacy of antiseptic molecules

The in vitro efficacy of locally applied antiseptic molecules against staphylococcal biofilm is frequently assessed by a set of standard quantitative and semi-quantitative methods. The development of software for parametric image processing allowed to obtain parametric data also from microscopic images of biofilm dyed with a variety of dyes, especially with propidium iodine and SYTO-9, differentiating dead from live cells. In this work, using confocal/epifluorescent microscopy, we analyzed such major properties of staphylococcal biofilm in vitro as its thickness, cellular density and share of Live/Dead cells within its individual parts. We also scrutinized the impact of sample preparation and antiseptic introduction on the outcome obtained. As a result of our analyses we developed a revelatory method of assessment of the impact of antiseptic agents on staphylococcal biofilm in vitro, in which the microscopic images are processed with the use of ABE formula (Antiseptics Biofilm Eradication) which implements all the data and phenomena detected and revealed within the course of this study. We tested ABE with regard to polyhexanide, povidone-iodine and hypochlorous antiseptics and found a high correlation between this parameter and the results obtained by means of traditional techniques. Taking into account the fact that in vitro results of the efficacy of antiseptic agents against staphylococcal biofilm are frequently applied to back up their use in hospitals and ambulatory units, our work should be considered an important tool providing reliable, parametric data with this regard.

Serotonin 1B receptor density mapping of the human brainstem using positron emission tomography and autoradiography

The serotonin 1B (5-HT1B) receptor has lately received considerable interest in relation to psychiatric and neurological diseases, partly due to findings based on quantification using Positron Emission Tomography (PET). Although the brainstem is an important structure in this regard, PET radioligand binding quantification in brainstem areas often shows poor reliability. This study aims to improve PET quantification of 5-HT1B receptor binding in the brainstem. Volumes of interest (VOIs) were selected based on a 3D [3H]AZ10419369 Autoradiography brainstem model, which visualized 5-HT1B receptor distribution in high resolution. Two previously developed VOI delineation methods were tested and compared to a conventional manual method. For a method based on template data, a [11C]AZ10419369 PET template was created by averaging parametric binding potential (BPND) images of 52 healthy subjects. VOIs were generated based on a predefined volume and BPND thresholding and subsequently applied to test-retest [11C]AZ10419369 parametric BPND images of 8 healthy subjects. For a method based on individual subject data, VOIs were generated directly on each individual parametric image. Both methods showed improved reliability compared to a conventional manual VOI. The VOIs created with [11C]AZ10419369 template data can be automatically applied to future PET studies measuring 5-HT1B receptor binding in the brainstem.

Morpho‐metabotyping the oxidative stress response

Oxidative stress and reactive oxygen species (ROS) are central to many physiological and pathophysiological processes. However, due to multiple technical challenges, it is hard to capture a comprehensive readout of the cell, involving both biochemical and functional status. We addressed this problem by developing a fully parallelized workflow for metabolomics (providing absolute quantities for > 100 metabolites including TCA cycle, pentose phosphate pathway, purine metabolism, glutathione metabolism, cysteine and methionine metabolism, glycolysis and gluconeogenesis) and live cell imaging microscopy. The correlative imaging strategy was applied to study morphological and metabolic adaptation of cancer cells upon short-term hydrogen peroxide (H2O2) exposure in vitro. The combination provided rich metabolic information at the endpoint of exposure together with imaging of mitochondrial effects. As a response, superoxide concentrations were elevated with a strong mitochondrial localization, and multi-parametric image analysis revealed a shift towards fragmentation. In line with this, metabolism reflected both the impaired mitochondrial function and shifts to support the first-line cellular defense and compensate for energy loss. The presented workflow combining high-end technologies demonstrates the applicability for the study of short-term oxidative stress, but it can be suitable for the in-depth study of various short-term oxidative and other cellular stress-related phenomena.

Software for Non-Parametric Image Registration of 2-Photon Imaging Data

Functional 2-photon microscopy is a key technology for imaging neuronal activity which can, however, contain non-rigid movement artifacts. Despite the established performance of variational optical flow (OF) estimation in different computer vision areas and the importance of movement correction for 2-photon applications, no OF-based method for 2-photon imaging is available. We developed the easy-to-use toolbox Flow-Registration that outperforms previous alignment tools and allows to align and reconstruct even low signal-to-noise 2-photon imaging data.

Resolved spectral variations of the centimetre-wavelength continuum from the ρ Oph W photo-dissociation-region

Cm-wavelength radio continuum emission in excess of free-free, synchrotron and Rayleigh-Jeans dust emission (excess microwave emission, EME), and often called ‘anomalous microwave emission’, is bright in molecular cloud regions exposed to UV radiation, i.e. in photo-dissociation regions (PDRs). The EME correlates with IR dust emission on degree angular scales. Resolved observations of well-studied PDRs are needed to compare the spectral variations of the cm-continuum with tracers of physical conditions and of the dust grain population. The EME is particularly bright in the regions of the ρ Ophiuchi molecular cloud (ρ Oph) that surround the earliest type star in the complex, HD 147889, where the peak signal stems from the filament known as the ρ Oph-W PDR. Here we report on ATCA observations of ρ Oph-W that resolve the width of the filament. We recover extended emission using a variant of non-parametric image synthesis performed in the sky plane. The multi-frequency 17 GHz to 39 GHz mosaics reveal spectral variations in the cm-wavelength continuum. At ∼30 arcsec resolutions, the 17-20 GHz intensities follow tightly the mid-IR, Icm∝I(8 μm), despite the breakdown of this correlation on larger scales. However, while the 33-39 GHz filament is parallel to IRAC 8 μm, it is offset by 15–20 arcsec towards the UV source. Such morphological differences in frequency reflect spectral variations, which we quantify spectroscopically as a sharp and steepening high-frequency cutoff, interpreted in terms of the spinning dust emission mechanism as a minimum grain size acutoff ∼ 6 ± 1 Å that increases deeper into the PDR.

Kinetic analysis of HER2-binding ABY-025 Affibody molecule using dynamic PET in patients with metastatic breast cancer

Background High expression of human epidermal growth factor receptor type 2 (HER2) represents an aggressive subtype of breast cancer. Anti-HER2 treatment requires a theragnostic approach wherein sufficiently high receptor expression in biopsy material is mandatory. Heterogeneity and discordance of HER2 expression between primary tumour and metastases, as well as within a lesion, present a complication for the treatment and require multiple biopsies. Molecular imaging using the HER2-targeting Affibody peptide ABY-025 radiolabelled with 68Ga-gallium for PET/CT is currently under investigation as a non-invasive tool for whole-body evaluation of metastatic HER2 expression. Initial studies demonstrated a high correlation between 68Ga-ABY-025 standardized uptake values (SUVs) and histopathology. However, detecting small liver lesions might be compromised by high background uptake. This study aimed to explore the applicability of kinetic modelling and parametric image analysis for absolute quantification of 68Ga-ABY-025 uptake and HER2-receptor expression and how that relates to static SUVs. Methods Dynamic 68Ga-ABY-025 PET of the upper abdomen was performed 0-45 min post-injection in 16 patients with metastatic breast cancer. Five patients underwent two examinations to test reproducibility. Parametric images of tracer delivery (K1) and irreversible binding (Ki) were created with an irreversible two-tissue compartment model and Patlak graphical analysis using an image-derived input function from the descending aorta. A volume of interest (VOI)-based analysis was performed to validate parametric images. SUVs were calculated from 2 h and 4 h post-injection static whole-body images and compared to Ki. Results Characterization of HER2 expression in smaller liver metastases was improved using parametric images. Ki values from parametric images agreed very well with VOI-based gold standard (R2 > 0.99, p < 0.001). SUVs of metastases at 2 h and 4 h post-injection were highly correlated with Ki values from both the two-tissue compartment model and Patlak method (R2 = 0.87 and 0.95, both p < 0.001). 68Ga-ABY-025 PET yielded high test-retest reliability (relative repeatability coefficient for Patlak 30% and for the two-tissue compartment model 47%). Conclusion 68Ga-ABY-025 binding in HER2-positive metastases was well characterized by irreversible two-tissue compartment model wherein Ki highly correlated with SUVs at 2 and 4 h. Dynamic scanning with parametric image formation can be used to evaluate metastatic HER2 expression accurately.