A study of Docetaxel-induced effects in MCF-7 cells by means of Raman microspectroscopy

Chemotherapies feature a low success rate of about 25%, and therefore, the choice of the most effective cytostatic drug for the individual patient and monitoring the efficiency of an ongoing chemotherapy are important steps towards personalized therapy. Thereby, an objective method able to differentiate between treated and untreated cancer cells would be essential. In this study, we provide molecular insights into Docetaxel-induced effects in MCF-7 cells, as a model system for adenocarcinoma, by means of Raman microspectroscopy combined with powerful chemometric methods. The analysis of the Raman data is divided into two steps. In the first part, the morphology of cell organelles, e.g. the cell nucleus has been visualized by analysing the Raman spectra with k-means cluster analysis and artificial neural networks and compared to the histopathologic gold standard method hematoxylin and eosin staining. This comparison showed that Raman microscopy is capable of displaying the cell morphology; however, this is in contrast to hematoxylin and eosin staining label free and can therefore be applied potentially in vivo. Because Docetaxel is a drug acting within the cell nucleus, Raman spectra originating from the cell nucleus region were further investigated in a next step. Thereby we were able to differentiate treated from untreated MCF-7 cells and to quantify the cell–drug response by utilizing linear discriminant analysis models

Functional real-time optoacoustic imaging of middle cerebral artery occlusion in mice.

Background and purposeLongitudinal functional imaging studies of stroke are key in identifying the disease progression and possible therapeutic interventions. Here we investigate the applicability of real-time functional optoacoustic imaging for monitoring of stroke progression in the whole brain of living animals.Materials and methodsThe middle cerebral artery occlusion (MCAO) was used to model stroke in mice, which were imaged preoperatively and the occlusion was kept in place for 60 minutes, after which optoacoustic scans were taken at several time points.ResultsPost ischemia an asymmetry of deoxygenated hemoglobin in the brain was observed as a region of hypoxia in the hemisphere affected by the ischemic event. Furthermore, we were able to visualize the penumbra in-vivo as a localized hemodynamically-compromised area adjacent to the region of stroke-induced perfusion deficit.ConclusionThe intrinsic sensitivity of the new imaging approach to functional blood parameters, in combination with real time operation and high spatial resolution in deep living tissues, may see it become a valuable and unique tool in the development and monitoring of treatments aimed at suspending the spread of an infarct area

Whole-brain optoacoustic images of a mouse.

<p>A: Structural features visible in the cryosection (right) are matched with the corresponding optoacoustic image features (left). The representative stained cryosection shows the infarct area caused by 1 h MCAO followed by 24 h reperfusion. In the optoacoustic image, the area corresponding to the brain is enclosed by a green dotted line. The area affected by MCAO has been enclosed by a yellow dotted line. B: the multispectral data unmixed for the oxygenated and deoxygenated hemoglobin are overlaid on the single wavelength image from above (790 nm) in blue and red, respectively; the sagittal sinus is highly oxygenated due to the oxygen-isoflurane anesthetic. C: A coronal-slice set from a representative animal (distance between slices: 1 mm) with the multispectral unmixed data for deoxygenated hemoglobin in the brain presented in blue. The post ischemia set shows clear asymmetry (green arrow), while symmetry is intact in the pre ischemia set. Red and green squares in A and C indicate the ROIs for the ratio analysis.</p

Deoxygenated hemoglobin distribution in several mice.

<p>From top down: Pre-, during, and 24 hours post-ischemia MSOT images for three MCAO mice (left to right) with corresponding H&E stained cryosections for each mouse (bottom; per mouse). MSOT: multispectral data for deoxygenated hemoglobin are presented in blue. Compared with the pre-ischemia situation, images taken during and 24 h post ischemia show clear asymmetry in the vicinity of the infarcted regions, as also apparent in the corresponding histological sections.</p

CBV ratio analysis for all the 7 imaged animals based on single wavelength optoacoustic images acquired at isosbestic wavelength of blood at 790 nm.

<p>Ratios of the mean in the ischemic cortex to the mean of its contralateral counterpart were calculated for the cortical areas (marked by red squares in Figs. 2A and 2C) and striatum areas (marked by green squares in Figs. 2A and 2C) and % changes relative to pre-ischemia values are presented. A: All mice show reduction in CBV in the cortex, followed by return to normal values after reperfusion at 24 h (middle). B: For the striatum areas, the mice show an increase of CBV during ischemia, which remains elevated in most of the animals after reperfusion. Averaged data of absolute ratio values from all the mice (box plots with median and 25^th and 75^th percentile) are shown in panels C and D for the cortical and striatum areas, respectively.</p

Illustrations of the procedure and experimental setup.

<p>A: Schematic representation of the MCAO procedure. The filament is forwarded into the MCA (1) through the ICA (3), starting at the bifurcation of the CCA (5) into the ECA (4) and ICA. This causes formation of the stroke volume (2). B: Schematic representation of the MSOT scanner. The mouse holder (1) secures the mouse (2) in the scanner and anesthesia is supplied through the gas mask (3). Optoacoustic signals are generated in the illumination plane (4) and detected by the transducer array (5), covering and angle of 172°.</p

Highly sensitive and specific detection of E. coli by a SERS nanobiosensor chip utilizing metallic nanosculptured thin films

A nanobiosensor chip, utilizing surface enhanced Raman spectroscopy (SERS) on nanosculptured thin films (nSTFs) of silver, was shown to detect Escherichia coli (E. coli) bacteria down to the concentration level of a single bacterium. The sensor utilizes highly enhanced plasmonic nSTFs of silver on a silicon platform for the enhancement of Raman bands as checked with adsorbed 4-aminothiophenol molecules. T-4 bacteriophages were immobilized on the aforementioned surface of the chip for the specific capture of target E. coli bacteria. To demonstrate that no significant non-specific immobilization of other bacteria occurs, three different, additional bacterial strains, Chromobacterium violaceum, Paracoccus denitrificans and Pseudomonas aeruginosa were used. Furthermore, experiments performed on an additional strain of E. coli to address the specificity and reusability of the sensor showed that the sensor operates for different strains of E. coli and is reusable. Time resolved phase contrast microscopy of the E. coli-T4 bacteriophage chip was performed to study its interaction with bacteria over time. Results showed that the present sensor performs a fast, accurate and stable detection of E. coli with ultra-small concentrations of bacteria down to the level of a single bacterium in 10 μl volume of the sample