Search

Abstract: Extremely brilliant infrared (IR) beams provided by synchrotron radiation sources are now routinely used in many facilities with available commercial spectrometers coupled to IR microscopes. Using these intense non-thermal sources, a brilliance two or three order of magnitude higher than a conventional source is achievable through small pinholes (<10μm) with a high signal to-noise ratio. IR spectroscopy is a powerful technique to investigate biological systems and offers many new imaging opportunities. The field of infrared biological imaging covers a wide range of fundamental issues and applied researches such as cell imaging or tissue imaging. Molecular maps with a spatial resolution down to the diffraction limit may be now obtained with a synchrotron radiation IR source also on thick samples. Moreover, changes of the protein structure are detectable in an IR spectrum and cellular molecular markers can be identified and used to recognize a pathological status of a tissue. Molecular structure and functions are strongly correlated and this aspect is particularly relevant for imaging. We will show that the brilliance of synchrotron radiation IR sources may enhance the sensitivity of a molecular signal obtained from small biosamples, e.g., a single cell, containing extremely small amounts of organic matter. We will also show that SR IR sources allow to study chemical composition and to identify the distribution of organic molecules in cells at submicron resolution is possible with a high signal-to-noise ratio. Moreover, the recent availability of two-dimensional IR detectors promises to push forward imaging capabilities in the time domain. Indeed, with a high current synchrotron radiation facility and a Focal Plane Array the chemical imaging of individual cells can be obtained in a few minutes. Within this framework important results are expected in the next years using synchrotron radiation and Free Electron Laser (FEL) sources for spectro-microscopy and spectral-imaging, alone or in combination with Scanning Near-field Optical Microscopy methods to study the molecular composition and dynamic changes in samples of biomedical interest at micrometric and submicrometric scales, respectively. [Copyright &y& Elsevier]

Abstract: We recently developed an atomic force microscopy–based protocol to use the roughness of the plasma membrane of erythrocytes (red blood cells, RBCs) as a morphological parameter, independently from the cell shape, to investigate the membrane-skeleton integrity in healthy and pathological cells. Here we apply the method to investigate a complex physiological phenomenon, the RBCs aging, that plays a major role in the regulation of the RBCs'' turnover. The aging, monitored morphologically and biochemically, has been accelerated and modulated by preventing oxidative stresses as well as the effects of proteases and divalent cations, and by artificially consuming the intracellular adenosine triphosphate. The collected data evidence that the progression of aging causes a drastic decrease of the measured roughness that is diagnostic of a progressive, adenosine triphosphate–dependent alteration of the membrane-skeleton properties. Finally, the degree of reversibility of such effects has been investigated as a function of aging time, enabling the detection of irreversible transformation in the RBCs'' structure and metabolism. From the Clinical Editor: An Atomic Force Microscopy-based protocol that uses the roughness of plasma membrane of erythrocytes as a morphological parameter to investigate membrane-skeleton integrity was applied to investigate red blood cell aging. The authors demonstrate that aging causes a decrease of the measured roughness, correlating with a progressive, ATP-dependent alteration of membrane-skeleton properties. [Copyright &y& Elsevier]

The advent of scanning near-field optical microscopy (SNOM) has augmented at the microscopic level the usefulness of IR spectroscopy. Two-dimensional imaging of chemical constituents makes this a very attractive and powerful new approach. In this paper we present SNOM results on boron-doped silicon and on biological growth medium by means of shear-force, reflectivity and photocurrent measurements. Such experiments allowed us to identify boron clusters embedded in silicon and the distribution of growth medium constituents with a lateral resolution well below the diffraction limit. [Copyright &y& Elsevier]

Abstract: Human erythrocytes (RBCs), stored at 4°C under nominal absence of external energy sources and calcium ions, show a gradual decrease in membrane roughness (R rms) at the end of which the appearance of morphological phenomena (spicules, vesicles and spherocytes) is observed on the cell membrane, phenomena that can mainly be ascribed to the ATP-dependent disconnection of the cortical cytoskeleton from the lipid bilayer. After depletion of the intracellular energy sources obtained under the extreme conditions chosen, treatment with a minimal rejuvenation solution makes the following remarks possible: (i) RBCs are able to regenerate adenosine triphosphate (ATP) and 2,3-bisphosphoglycerate only up to 4days of storage at 4°C, whereas from the eighth day energy stocks cannot be replenished because of a disorder in the transmembrane mechanisms of transport; (ii) the RBCs'' roughness may be restored to the initial value (i.e. that observed in fresh RBCs) only in samples stored up to 4–5days, whereas after the eighth day of storage the rejuvenation procedure appears to be inefficient; (iii) membrane physical properties – as measured by R rms – are actually controlled by the metabolic production of ATP, necessary to perform the RBCs'' basic functions; (iv) once energy stores cannot be replenished, a regulated sequence of the morphological events (represented by local buckles that lead to formation of spicules and vesicles of the lipid bilayer with generation of spherocytes) is reminiscent of the RBCs'' apoptotic final stages; (v) the morphological phenomenology of the final apoptotic stages is passive (i.e. determined by simple mechanical forces) and encoded in the mechanical properties of the membrane-skeleton; and (vi) necrotic aspects (e.g. disruption of cell membrane integrity, so that intracellular protein content is easily released) ensue when RBCs are almost totally (≥90%) depleted in an irreversible way of the energetic stores. [Copyright &y& Elsevier]

Abstract: A novel approach to the study of RBCs based on the collection of three-dimensional high-resolution AFM images and on the measure of the surface roughness of their plasma membrane is presented. The dependence of the roughness from several parameters of the imaging was investigated and a general rule for a trustful analysis and comparison has been suggested. The roughness of RBCs is a morphology-related parameter which has been shown to be characteristic of the single cells composing a sample, but independent of the overall geometric shape (discocyte or spherocyte) of the erythrocytes, thus providing extra-information with respect to a conventional morphology study. The use of the average roughness value as a label of a whole sample was tested on different kinds of samples. Analyzed data revealed that the quantitative roughness value does not change after treatment of RBCs with various commonly used fixation and staining methods while a drastic decrease occurs when studying cells with membrane–skeletal alteration both naturally occurring or artificially induced by chemical treatments. The present method provides a quantitative and powerful tool for a novel approach to the study of erythrocytes structure through an ultrastructural morphological analysis with the potential to give information, in a non-invasive way, on the RBCs function. [Copyright &y& Elsevier]

Abstract: Lipids are the principal components of biologically relevant structures as cellular membranes. They have been the subject of many studies due to their biological relevance and their potential applications. Different techniques, such as Langmuir–Blodgett and vesicle-fusion deposition, are available to deposit ordered lipid films on etched surfaces. Recently, a new technique of lipid film deposition has been proposed in which stacks of a small and well-controlled number of bilayers are prepared on a suitable substrate using a spin-coater. We studied the morphological properties of multi-layers made of cationic and neutral lipids (DOTAP and DOPC) and mixtures of them using dynamic mode atomic force microscopy (AFM). After adapting and optimizing, the spin-coating technique to deposit lipids on a chemically etched Silicon (1,0,0) substrate, a morphological nanometer-scale characterization of the aforementioned samples has been provided. The AFM study showed that an initial layer of ordered vesicles is formed and, afterward, depending on details of the spin-coating preparation protocol and to the dimension of the silicon substrate, vesicle fusion and structural rearrangements of the lipid layers may occur. The present data disclose the possibility to control the lipid''s structures by acting on spin-coating parameters with promising perspectives for novel applications of lipid films. [Copyright &y& Elsevier]