Slávka Kaščáková

Detection of an estrogen derivative in two breast cancer cell lines using a single core multimodal probe for imaging (SCoMPI) imaged by a panel of luminescent and vibrational techniques

3-Methoxy-17α-ethynylestradiol or mestranol is a prodrug for ethynylestradiol and the estrogen component of some oral contraceptive formulations. We demonstrate here that a single core multimodal probe for imaging - SCoMPI - can be efficiently grafted onto mestranol allowing its tracking in two breast cancer cell lines, MDA-MB-231 and MCF-7 fixed cells. Correlative imaging studies based on luminescence (synchrotron UV spectromicroscopy, wide field and confocal fluorescence microscopies) and vibrational (AFMIR, synchrotron FTIR spectromicroscopy, synchrotron-based multiple beam FTIR imaging, confocal Raman microspectroscopy) spectroscopies were consistent with one another and showed a Golgi apparatus distribution of the SCoMPI-mestranol conjugate in both cell lines.

Fluorescence Spectroscopic Study of Hypericin‐photosensitized Oxidation of Low‐density Lipoproteins

Abstract By means of UV-VIS absorption and fluorescence spectroscopy, we demonstrate that the photosensitizer hypericin (Hyp) interacts nonspecifically with low-density lipoproteins (LDL), most probably with the lipid fraction of LDL. The molar ratio of monomeric Hyp binding to nonoxidized LDL and mildly oxidized LDL is 30:1. Increasing the Hyp concentration further leads to the formation of Hyp aggregates inside the LDL molecule. We also demonstrate that photoactivated Hyp oxidizes LDL in a light dose and excitation wavelength dependent manner. The level of oxidation of LDL depends on the amount of Hyp inside the LDL molecule. The maximum of the photosensitized oxidation of the LDL by Hyp is achieved for a 30:1 molar ratio, which corresponds to the maximum concentration of monomeric form of Hyp in LDL.

Photosensitizer effects on cancerous cells: A combined study using synchrotron infrared and fluorescence microscopies

Hypocrellin A (HA), a lipid-soluble peryloquinone derivative, isolated from natural fungus sacs of Hypocrella bambusae, has been reported to be a highly potential photosensitizer in photodynamic therapy (PDT). It has been studied increasingly because of its anticancer activities when irradiated with light. We have studied the interaction mechanisms of HA with HeLa cells as a function of incubation time. Fluorescence microscopy confirmed that HA localisation is limited in the cytoplasm before eventually concentrating in clusters around the nucleus. The IR spectra of HA-treated, PDT-treated and control HeLa cells were recorded at the ESRF Infrared beamline (ID21). Principal component analysis has been used to assess the IR spectral changes between the various HeLa cells spectral data sets (The Unscrambler software, CAMO). PCA revealed that there is a frequency shift of protein amide I and amide II vibrational bands, indicating changes in the protein secondary structures of the HA-treated and PDT-treated cancer cells compared to the control cells. In addition, the relative DNA intensity in HA-treated cells decreases gradually along the incubation time. The use of synchrotron infrared microscopy is shown to be of paramount importance for targeting specifically the biochemical modification induced in the cell nucleus.

Tryptophan-functionalized gold nanoparticles for deep UV imaging of microbial cells

Biocompatible fluorescent nanostructures were prepared by a functionalization of gold nanoparticles with the amino acid tryptophan. The gold-tryptophan bioconjugates were investigated by TEM and HRTEM and various spectroscopy methods (XPS, FTIR, UV-vis and photoluminescence). It was found that the gold nanoparticles, initially 8 nm in diameter, aggregate in the presence of the amino acid. From the XPS and FTIR spectroscopy results, it was concluded that the tryptophan gold interactions mainly take place via indole and carboxyl groups. Although the indole group is involved in the interaction with the gold surfaces, the tryptophan-gold hybrids showed strong fluorescence due to the presence of multilayers of tryptophan. Deep ultra violet (DUV) imaging performed at the SOLEIL synchrotron showed that it is possible to detect these hybrid nanostructures within Escherichia coli cells.

Discrimination of cirrhotic nodules, dysplastic lesions and hepatocellular carcinoma by their vibrational signature

BackgroundHepatocarcinogenesis is a multistep process characterized in patients with chronic liver diseases by a spectrum of hepatic nodules that mark the progression from regenerative nodules to dysplastic lesions followed by hepatocellular carcinoma (HCC). The differential diagnosis between precancerous dysplastic nodules and early HCC still represents a challenge for both radiologists and pathologists. We addressed the potential of Fourier transform-infrared (FTIR) microspectroscopy for grading cirrhotic nodules on frozen tissue sections.MethodsThe study was focused on 39 surgical specimens including normal livers (nxa0=xa011), dysplastic nodules (nxa0=xa06), early HCC (nxa0=xa01), progressed HCC on alcoholic cirrhosis (nxa0=xa010) or hepatitis C virus cirrhosis (nxa0=xa011). The use of the bright infrared source emitted by the synchrotron radiation allowed investigating the biochemical composition at the cellular level. Chemical mapping on whole tissue sections was further performed using a FTIR microscope equipped with a laboratory-based infrared source. The variance was addressed by principal component analysis.ResultsProfound alterations of the biochemical composition of the pathological liver were demonstrated by FTIR microspectroscopy. Indeed, dramatic changes were observed in lipids, proteins and sugars highlighting the metabolic reprogramming in carcinogenesis. Quantifiable spectral markers were characterized by calculating ratios of areas under specific bands along the infrared spectrum. These markers allowed the discrimination of cirrhotic nodules, dysplastic lesions and HCC. Finally, the spectral markers can be measured using a laboratory FTIR microscope that may be easily implemented at the hospital.ConclusionMetabolic reprogramming in liver carcinogenesis can constitute a signature easily detectable using FTIR microspectroscopy for the diagnosis of precancerous and cancerous lesions.

Quantitative Assessment of Liver Steatosis on Tissue Section Using Infrared Spectroscopy

© 2015 by the AGA Institute 0016-5085/

Rapid and reliable diagnosis of Wilson disease using X-ray fluorescence

36.00 http://dx.doi.org/10.1053/j.gastro.2014.11.038 Ltreatment of patients with cirrhosis or hepatocellular carcinoma. Due to the shortage of grafts, transplantation teams are using marginal grafts from expanded criteria donors. These marginal grafts constitute an additional source of organs but also an increased risk factor of primary nonfunction or dysfunction of the graft mainly due to their poor quality. Steatosis is one of the most important factors affecting liver allograft function, mostly because of more severe ischemia-reperfusion injury. More than 30% of the population in Western countries is estimated to exhibit this lesion. Steatosis is characterized by the intra-cellular accumulation of triacylglycerides (TG) resulting in the formation of lipid vesicles in the hepatocytes. The gold standard to assess hepatic steatosis in liver grafts during the transplantation procedure is the histologic examination of frozen sections by a pathologist. The quantitative histologic evaluation of steatosis is based on the percentage of hepatocytes containing cytoplasm fat inclusions. Most transplant surgeons currently discard grafts when the total amount of hepatic steatosis is more than 60% because of an expected high risk of graft failure. However, while there is general agreement that mild steatosis (<30%) causes minor graft injury, the use of grafts with moderate steatosis (30%-60%) remains a challenging issue with primary non-function rates ranging between 0% and 75% after LT. The major issue is that assessment of hepatic steatosis on histologic sections is an imperfect and not reproducible method. A recent study demonstrated poor inter-observer agreement among expert pathologists for quantitative assessment of steatosis on paraffin sections. Despite improvements using stereology, the emergency timing of liver transplantation imposes a rapid process such as the use of frozen sections leading to some damages in the tissue morphology and thus to more imprecise assessment of steatosis (Figure 1A). Finally, we have demonstrated that the histological estimation of steatosis is poorly correlated with the true lipid content of the liver tissue after lipid extraction from the same surgical specimen followed by gas phase chromatography coupled to mass spectrometry (GC-MS) (Figure 1B). Thus, an alternative method to histopathology providing an objective assessment of steatosis is an unmet need.

Interaction of amino acid-functionalized silver nanoparticles and Candida albicans polymorphs: A deep‐UV fluorescence imaging study

Wilsons disease (WD) is a rare autosomal recessive disease due to mutations of the gene encoding the copper‐transporter ATP7B. The diagnosis is hampered by the variability of symptoms induced by copper accumulation, the inconstancy of the pathognomonic signs and the absence of a reliable diagnostic test. We investigated the diagnostic potential of X‐ray fluorescence (XRF) that allows quantitative analysis of multiple elements. Studies were performed on animal models using Wistar rats (nu2009=u200910) and Long Evans Cinnamon (LEC) rats (nu2009=u200911), and on human samples including normal livers (nu2009=u200910), alcohol cirrhosis (nu2009=u20098), haemochromatosis (nu2009=u200910), cholestasis (nu2009=u20096) and WD (nu2009=u200922). XRF experiments were first performed using synchrotron radiation to address the elemental composition at the cellular level. High‐resolution mapping of tissue sections allowed measurement of the intensity and the distribution of copper, iron and zinc while preserving the morphology. Investigations were further conducted using a laboratory X‐ray source for irradiating whole pieces of tissue. The sensitivity of XRF was highlighted by the discrimination of LEC rats from wild type even under a regimen using copper deficient food. XRF on whole formalin‐fixed paraffin embedded needle biopsies allowed profiling of the elements in a few minutes. The intensity of copper related to iron and zinc significantly discriminated WD from other genetic or chronic liver diseases with 97.6% specificity and 100% sensitivity. This study established a definite diagnosis of Wilsons disease based on XRF. This rapid and versatile method can be easily implemented in a clinical setting.

Single vs. two-photon microscopy for label free intrinsic tissue studies in the UV light region

The interaction of the tryptophan functionalized Ag nanoparticles and live Candida albicans cells was studied by synchrotron excitation deep-ultraviolet (DUV) fluorescence imaging at the DISCO beamline of Synchrotron SOLEIL. DUV imaging showed that incubation of the fungus with functionalized nanoparticles results in significant increase in the fluorescence signal. The analysis of the images revealed that the interaction of the nanoparticles with (pseudo)hyphae polymorphs of the diploid fungus was less pronounced than in the case of yeast cells or budding spores. The changes in the intensity of the fluorescence signals of the cells after incubation were followed in [327-353nm] and [370-410nm] spectral ranges that correspond to the fluorescence of tryptophan in non-polar and polar environment, respectively. As a consequence of the environmental sensitivity of the silver-tryptophan fluorescent nanoprobe, we were able to determine the possible accumulation sites of the nanoparticles. The analysis of the intensity decay kinetics showed that the photobleaching effects were more pronounced in the case of the functionalized nanoparticle treated cells. The results of time-integrated emission in the mentioned spectral ranges suggested that the nanoparticles penetrate the cells, but that the majority of the nanoparticles attach to the cells surfaces.

DUV Autofluorescence Microscopy for Cell Biology and Tissue Histology Biology of the Cell Biology of the Cell

Fibrillar distribution in the rat tail tendon and mice liver can be measured using optical methods. Two-photon excitation provides easy assessment of fibrotic collagen types I and II. Single photon deep ultraviolet (DUV) excitation imaging highlights all collagen types without discrimination. Their combination on the same tissue area provides a better overview of collagens in fibrillar diseases.