Evidence for Hoogsteen GC Base Pairs in the Proton-Induced Transition from Right-Handed to Left-Handed Poly(dG-dC) . Poly(dG-dC)

The structure of double-helical poly(dG-dC)·poly(dG-dC) is investigated at various pH values with Raman spectroscopy, absorption spectroscopy, and circular dichroism. A comparison is made between the B-form with Watson−Crick base pairing at 1 mM [Na+] and pH 7.2, the Z-form with Watson−Crick base pairing at 4 M [Na+] and pH 7.2, and a different structure at 1 mM [Na+] and pH 4.5 as well as at 150 mM [Na+] and pH 3.1. The CD spectrum of poly(dG-dC)·poly(dG-dC) under the latter conditions does not show a negative band at 290 nm. The structure is a double-helical structure different from the B-form and the Z-form according to circular dichroism, Raman, and absorption spectroscopic studies. The Raman spectra evidence that the structure contains Hoogsteen base pairing. This can be accommodated in the double helix when the cytosine group is protonated and the sugar−guanine conformer has adopted a C2‘-endo/syn conformation. It is shown that this antiparallel-stranded Hoogsteen base paired structure can be maintained under varying conditions, balancing the decrease in pH with an increased salt concentration. It is further concluded that the proton-induced transition from a Watson−Crick to a Hoogsteen base pair is aided by a decrease of [Na+] at pH 4.5 and occurs prior to a conversion from a right-handed helix to a left-handed heli

Axial resolution of confocal Raman microscopes: Gaussian beam theory and practice

A straightforward and transparent model, based on Gaussian beam optics, for the axial r0 resolution of a confocal microscope is presented. A confocal Raman microscope was used to determine the axial confocality in practice. The axial response of a thin planar object was measured for three different objectives, two pinhole sizes and a slit. The results show that, in the case of a confocal configuration, the response calculated with the model provides a good prediction of the axial resolution of the confocal microscope

Application of Raman Microspectroscopic and Raman imaging techniques for cell biological studies

Raman spectroscopy is being used to study biological molecules for some three decades now. Thanks to continuing advances in instrumentation more and more applications have become feasible in which molecules are studied in situ, and this has enabled Raman spectroscopy to enter the realms of biomedicine and cell biology [1-5].\ud Here we will describe some of the recent work carried out in our laboratory, concerning studies of human white blood cells and further instrumentational developments

Localization Study of Co-Phthalocyanines in Cells by Raman Micro(spectro)scopy

An investigation of intracellular localization of Co-phthalocyanines is reported. The Raman images of K562 cells stained with phthalocyanine were acquired. To understand the peculiarities of the Raman images, measurements were performed at different z-axis positions. The intracellular concentration of phthalocyanine was estimated. A colocalization study was carried out using the fluorescence probes FITC-dextran and acridine orange by means of Raman and fluorescence microscopy. Partial colocalization with both probes was revealed