Papers by Francesca Bertani
Journal of Biophotonics, 2015
A novel hyperspectral confocal microscopy method to separate different cell populations in a co-c... more 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).
Blood, 2008
Microbial products, including lipopolysaccharide (LPS), an agonist of Toll-like receptor 4 (TLR4)... more Microbial products, including lipopolysaccharide (LPS), an agonist of Toll-like receptor 4 (TLR4), regulate the lifespan of dendritic cells (DCs) by largely undefined mechanisms. Here, we identify a role for calcium-calmodulin-dependent kinase IV (CaMKIV) in this survival program. The pharmacologic inhibition of CaMKs as well as ectopic expression of kinase-inactive CaMKIV decrease the viability of monocyte-derived DCs exposed to bacterial LPS. The defect in TLR4 signaling includes a failure to accumulate the phosphorylated form of the cAMP response element-binding protein (pCREB), Bcl-2, and Bcl-xL. CaMKIV null mice have a decreased number of DCs in lymphoid tissues and fail to accumulate mature DCs in spleen on in vivo exposure to LPS. Although isolated Camk4-/- DCs are able to acquire the phenotype typical of mature cells and release normal amounts of cytokines in response to LPS, they fail to accumulate pCREB, Bcl-2, and Bcl-xL and therefore do not survive. The transgenic expression of Bcl-2 in CaMKIV null mice results in full recovery of DC survival in response to LPS. These results reveal a novel link between TLR4 and a calcium-dependent signaling cascade comprising CaMKIV-CREB-Bcl-2 that is essential for DC survival.
We report the development of a new optical imaging instrument and present one of the possible dif... more We report the development of a new optical imaging instrument and present one of the possible different applications. This hyperspectral reflectance confocal microscope has been designed and built within a national project called SKINTARGET, formerly approved by an IDEAS EU grant. As a confocal microscope, this instrument is capable of noninvasively imaging morphological features and high-resolution spectroscopic properties of surfaces but also inside thick specimens. Moreover, it collects both spatial and spectroscopic information from the sample by recording spectral reflectance response using as a probe white light from a supercontinuum laser source ranging from 450 to 2500nm. The aim is to realize a full spectral fingerprint of human sample tissues, observed in-vivo as well as in-vitro, correlating the 4-dimensional response to the pathological state of the sample. While the related activity of this project is ongoing, first data on tests sample has clearly shown the potential a...
The Analyst, 2015
Principal component analysis is applied to analyse the Raman maps collected on carbon nanotubes a... more Principal component analysis is applied to analyse the Raman maps collected on carbon nanotubes at different degrees of oxidation and functionalization with dye labeling molecules. The results are used to demonstrate that the technique is extremely effective in clustering data and comparing preparation protocols, so that it enables drawing of a fast and reliable classification of the molecule propensity to interact with pristine and oxidized carbon nanotubes. The spectral findings are supported and elucidated by several experimental techniques, thermogravimetry and steady-state and time-resolved fluorescence measurements, and by computational modeling, showing that the proposed methodology could represent a powerful and routine test for the rational design of functional nanostructures.
Biomedical Materials, 2015
Journal of the European Optical Society: Rapid Publications, 2013
Among the optical techniques used for exploring the properties of cells and tissues, those based ... more Among the optical techniques used for exploring the properties of cells and tissues, those based on hyperspectral label-free analysis are particularly interesting due to their non-invasive character and their ability to fast collect a huge number of information on the different sample constituents and their spatial distribution. Here we present results obtained with a novel hyperspectral reflectance confocal microscope of label-free discrimination of cells undergoing apoptosis. Our data, analyzed by means of a powerful statistical method, enable to obtain information on the biological status at a single cell level through the local measurement of reflectivity. Furthermore, an optical model of the local dielectric response gives an additional insight of the parameters linking the optical responsivity to the biological status.
The possibility to assess single ce l l characterization - including meta boli c state, pathologi... more The possibility to assess single ce l l characterization - including meta boli c state, pathological classification, and answer to a therapeutic treatment- through non invasive o ptical methods is gaining more importance every day. Here we present a novel methodological approach to obtain and evaluate VIS-NIR local reflectance spectra from cells in culture conditions without the use of any exogenous dye. We performed this preliminary measurements using a supercontinuum ultra wide range confocal microscope (1) and a white light laser as source light which allows to obtain both images and a complete VIS-NIR spectrum for each image pixel. We used this technique in two sets of experiments involving skin cells. In the first set we record ed spectra from in vitro keratinocytes cell culture undergoing UV induced apoptosis. Reflectance spectra averaged aver the enti re single cells have been registered in the 500-900 nm wavelength range at different time point, and showed remarkably differe...
AIP Advances, 2011
We report the design and implementation of a new reflectance laser scanning confocal system with ... more We report the design and implementation of a new reflectance laser scanning confocal system with spectroscopy imaging capabilities. Confocal spectroscopy is achieved by using a very broad spectral range supercontinuum source capable of high precision reflectance data in the VIS-IR spectral range thanks to an almost achromatic optical layout. With this apparatus we collect each single scanning point as a whole spectrum in a continuous range, associated with the optical section imaging possibilities typical of a confocal set up. While such a microscope has been developed for bio medical analysis of human skin and other similar applications, first test results on solid samples produce spectroscopic results that, compared to analytical models based on the Abelés matrix transfer methods, show a very good agreement, opening new possibilities of a complete spectroscopic fingerprinting of samples with microscopic details. Copyright 2011 Author(s). This article is distributed under a Creative Commons Attribution 3.0 Unported License.
![Research paper thumbnail of Novel fluorescent security marker. Part I: morphological and optical properties of 2-amino-6-ethoxy-4-[4-(4-morpholinyl)phenyl]-3,5-pyridinedicarbonitrile nanoparticles](https://attachments.academia-assets.com/42032137/thumbnails/1.jpg)
Journal of Nanoparticle Research, 2012
Newly synthesized fluorescent nanoparticles of 2-amino-6-ethoxy-4-[4-(4-morpholinyl)phenyl]-3, 5-... more Newly synthesized fluorescent nanoparticles of 2-amino-6-ethoxy-4-[4-(4-morpholinyl)phenyl]-3, 5-pyridinedicarbonitrile have been developed and characterized for possible applications as security marker in paper documents. Nanoparticles have been prepared by reprecipitation in water under sonication. The size and the shape of these nanoparticles, characterized by light scattering and atomic force microscopy, have been found to be highly dependent on sonication power. Typical sizes range from tens to hundreds of nanometers. Furthermore, a remarkable increase in the fluorescence yield has been observed as nanoparticles sizes decrease. Finally, all of the above features, together with the striking stability of optical and mechanical properties over the course of months, allow for straightforward applications that rely on strong and stable fluorescence such as marking important or valuable documents.
Journal of Cellular Physiology, 2008
Induction of cell death in HeLa cells with TNF and cycloheximide (CHX) required an adequate ATP s... more Induction of cell death in HeLa cells with TNF and cycloheximide (CHX) required an adequate ATP supply and was accompanied by decrease in intracellular pH, translocation of Bax, perinuclear clustering of the mitochondria, and cytochrome c release. The chloride channel inhibitor furosemide prevented the intracellular acidification, the translocation of Bax and the cell death. Cyclosporin A (CyA) or bongkrekic acid (BK) inhibited the induction of the MPT, the release of cytochrome c and the cell death without affecting the perinuclear clustering of the mitochondria or the translocation of Bax. Energy depletion with the ATP synthase inhibitor oligomycin or the uncoupler FCCP in the presence of 2-deoxy-glucose prevented the perinuclear clustering of the mitochondria and the cell killing. However, mitochondrial translocation of Bax was still observed. By contrast, cytochrome c was released in the oligomycin-treated cells but not in the same cells treated with FCCP. The data demonstrate that apoptosis in HeLa cells is ATP dependent and requires the translocation of Bax. The movement of Bax to the mitochondria occurs before and during the perinuclear clustering of these organelles and does not require the presence of ATP. The release of cytochrome c depends on the induction of the mitochondrial permeability transition but not ATP content.
Biophysical Journal, 2006
In early phases of atherogenesis, droplets and vesicles accumulate in the subendothelial extracel... more In early phases of atherogenesis, droplets and vesicles accumulate in the subendothelial extracellular space of arterial intima. There is much evidence to suggest that these droplets, ranging between 100 and 400 nm, derive from modified low-density lipoprotein (LDL). In investigations of the formation mechanism of these droplets, LDL fusion was previously induced in vitro by proteolysis, lipolysis, oxidation, and vigorous shaking, but all treatments failed to reproduce the size distribution range of in vivo droplets, mostly resulting, instead, in particles with a diameter intermediate between that of one and two LDL. Our approach was meant to mimic LDL aging in plasma. LDL isolated from plasma that was incubated overnight at 37°C is slightly modified in the secondary structure of its protein component and is primed to form very large aggregates according to a reaction-limited mechanism. This mechanism requires interactions between selected surface sites, whereas massive fusion is ruled out. In the frame of the general theory for colloids, the aggregation of LDL aged in plasma fulfills all the requirements of the reaction-limited mechanism, encompassing 1), exponential growth; 2), fractal structure, with the dimension of elementary constituent still consistent with a single LDL; and 3), extreme polydispersity of aggregates, with shape and dimension very close to that of droplets observed in vivo.
Blood, 2008
Microbial products, including lipopolysaccharide (LPS), an agonist of Toll-like receptor 4 (TLR4)... more Microbial products, including lipopolysaccharide (LPS), an agonist of Toll-like receptor 4 (TLR4), regulate the lifespan of dendritic cells (DCs) by largely undefined mechanisms. Here, we identify a role for calcium-calmodulin-dependent kinase IV (CaMKIV) in this survival program. The pharmacologic inhibition of CaMKs as well as ectopic expression of kinase-inactive CaMKIV decrease the viability of monocyte-derived DCs exposed to bacterial LPS. The defect in TLR4 signaling includes a failure to accumulate the phosphorylated form of the cAMP response element-binding protein (pCREB), Bcl-2, and Bcl-xL. CaMKIV null mice have a decreased number of DCs in lymphoid tissues and fail to accumulate mature DCs in spleen on in vivo exposure to LPS. Although isolated Camk4-/- DCs are able to acquire the phenotype typical of mature cells and release normal amounts of cytokines in response to LPS, they fail to accumulate pCREB, Bcl-2, and Bcl-xL and therefore do not survive. The transgenic expression of Bcl-2 in CaMKIV null mice results in full recovery of DC survival in response to LPS. These results reveal a novel link between TLR4 and a calcium-dependent signaling cascade comprising CaMKIV-CREB-Bcl-2 that is essential for DC survival.
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Papers by Francesca Bertani