Chemically resolved imaging of biological cells and thin films by infrared scanning near-field optical microscopy

The infrared (IR) absorption of a biological system can potentially report on fundamentally important microchemical properties. For example, molecular IR profiles are known to change during increases in metabolic flux, protein phosphorylation, or proteolytic cleavage. However, practical implementation of intracellular IR imaging has been problematic because the diffraction limit of conventional infrared microscopy results in low spatial resolution. We have overcome this limitation by using an IR spectroscopic version of scanning near-field optical microscopy (SNOM), in conjunction with a tunable free-electron laser source. The results presented here clearly reveal different chemical constituents in thin films and biological cells. The space distribution of specific chemical species was obtained by taking SNOM images at IR wavelengths (lambda) corresponding to stretch absorption bands of common biochemical bonds, such as the amide bond. In our SNOM implementation, this chemical sensitivity is combined with a lateral resolution of 0.1 micro m ( approximately lambda/70), well below the diffraction limit of standard infrared microscopy. The potential applications of this approach touch virtually every aspect of the life sciences and medical research, as well as problems in materials science, chemistry, physics, and environmental research.     

Frontal sinus fractures: guidelines to management

The appropriate management of frontal sinus fractures is controversial. Experience with 78 frontal sinus fractures over a 9-year period was reviewed, and the fractures were classified into anterior wall, anterobasilar, and frontal skull fracture extensions. The presence of a concomitant CSF leak or an air-fluid level in the sinus was a diagnostic clue of posterior wall involvement. Ablation or obliteration of the fractured frontal sinus is not necessary. Primary reconstruction of the sinus and nasofrontal duct drainage constitute the preferred treatment. "Cranialization" of a severely damaged sinus is performed by excision of the posterior wall plugging of the nasofrontal duct and reconstruction of the anterior wall. Reconstruction of the anterior wall with primary bone grafting may be necessary in selected patients.     

From phage display to magnetophage, a new tool for magnetic resonance molecular imaging

Phage display, an extremely promising technology in the context of molecular imaging, allows for the selection of peptides interacting with virtually any target from a heterogeneous mixture of bacteriophages. In this work, we propose the concept of magnetophages, obtained by covalent coupling of ultrasmall particles of iron oxide (USPIO) to the proteins of the phage wall. To validate magnetophages as a magnetic resonance imaging contrast agent (MRI), we have used as a prototype the clone E3 because of its specific affinity for phosphatidylserine, a marker of apoptosis. Enzyme-linked immunosorbent assay showed that E3 magnetophages incubated with phosphatidylserine retained the properties of the nonmagnetically labeled phages. The usefulness of magnetophages as an MRI contrast agent was estimated by incubation with phosphatidylcholine and phosphatidylserine or with apoptotic and control cells. Under these conditions, E3 magnetophages allow the discrimination of phosphatidylserine from phosphatidylcholine and of apoptotic cells from control ones. Injected in vivo, magnetophages are rapidly cleared from the blood stream and internalized by the phagocytic cells of the liver. To abrogate this problem, USPIO were pegylated to obtain stealthy E3-PEG-magnetophages, invisible to phagocytic cells, which were successfully targeted to apoptotic liver. If this feature demonstrated for E3 magnetophages can be extrapolated to other phage display selected entities, magnetophages become an original system which allows validation of the candidate binding peptides before their synthesis is considered. The concept of the magnetophage could be extended to other imaging modalities by replacing USPIO with an adequate reporter (i.e., radiolabeled phages).     

Comprehensive investigation of the non-covalent binding of MRI contrast agents with human serum albumin

Three techniques, electrospray mass spectrometry, ultrafiltration, and proton relaxometry, are compared in the context of the quantitative analysis of non-covalent binding between human serum albumin (HSA) and MRI contrast agents. The study of the affinity by proton relaxometry reveals the association constant and the number of interaction sites assuming that all sites are identical and independent. Ultrafiltration was adapted for the study of paramagnetic complexes. This technique confirmed the results obtained by relaxometry. Electrospray mass spectrometry, an original method able to study non-covalent binding because of its soft ionization process that allows for the survival of weak binding, provides qualitative and quantitative results. Electrospray mass spectrometry confirmed the affinity measured by proton relaxometry and ultrafiltration. This technique requires very small amounts of products and directly gives the stoichiometry of the association, information not easily obtained by classic techniques. Nevertheless, proton relaxometry remains a useful and mandatory technique for determining the enhancement of the relaxation subsequent to the binding although it demands large amounts of compounds. It is to be pointed out that even if the three techniques lead to a similar ranking of the affinity of the contrast agents for HSA, the absolute values of the association constants disagree as a result of the difference in the experimental conditions (presence of salt, native protein or desalted one, approximations in the fitting of the data, liquid or gas phases).