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The development of laser traps has made it possible to investigate single cells and record real-time Raman spectra during a heat-denaturation process when the temperature of the surrounding medium is increased. Large changes in the phenylalanine band (1004 cm-1) of near-infrared spectra between living and heat-treated cells were observed in yeast and Escerichia coli and Enterobacter aerogenes bacteria. This change appears to reflect the change in environment of phenylalanine as proteins within the cells unfold as a result of increasing temperatures. As a comparison, we measured Raman spectra of native and heat-denatured solutions of bovine serum albumin proteins, and a similar change in the phenylalanine band of spectra was observed. In addition, we measured Raman spectra of native and heat-treated solutions of pure phenylalanine molecules; no observable difference in vibrational spectra was observed. These findings may make it possible to study conformational changes in proteins within single cells. © 2003 American Institute of Physics. [ABSTRACT FROM AUTHOR]

A study on Chloroquine (CQ) accumulation in live malaria parasites performed at low nanomolar CQ concentrations is presented. The quantification of CQR via cytocidal CQ activity and examination of CQ accumulation at medically relevant [LD.sub.50] doses revealed that the reduced CQ accumulation is not the underlying cause of CQ resistance (CQR).

A NIR confocal laser-tweezers-Raman spectroscopy (LTRS) system is developed that combines the advantages of optical trapping, Raman spectroscopy, and the shifted excitation difference technique for characterization of single motile biological cells. This system effectively removes the strong fluorescence and stray light interference on the single cell Raman spectra and provides high sensitivity.

Counting ion channels on cell membranes is of fundamental importance for the study of channel biophysics. Channel counting has thus far been tackled by classical approaches, such as radioactive labeling of ion channels with blockers, gating current measurements, and nonstationary noise analysis. Here, we develop a counting method based on patch-clamp fluorometry (PCF), which enables simultaneous electrical and optical recordings, and apply it to EGFP-tagged, hyperpolarization-activated and cyclic nucleotide-regulated (HCN) channels. We use a well-characterized and homologous cyclic nucleotide-gated (CNG) channel to establish the relationship between macroscopic fluorescence intensity and the total number of channels. Subsequently, based on our estimate of the total number of HCN channels, we determine the single-channel conductance of HCN1 and HCN2 to be 0.46 and 1.71 pS, respectively. Such a small conductance would present a technical challenge for traditional electrophysiology. This PCF-based technique provides an alternative method for counting particles on cell membranes, which could be applied to biophysical studies of other membrane proteins. [ABSTRACT FROM AUTHOR]

We demonstrate the first reported use of single-walled carbon nanotubes as nano-optical probes in apertureless near-field fluorescence microscopy. We show that, in contrast to silicon probes, carbon nanotubes always cause strong fluorescence quenching when used to image dye-doped polystyrene spheres and Cd-Se quantum dots. For quantum dots, the carbon nanotubes induce very strong near-field contrast with a spatial resolution of 20 nm. Images of dye-doped spheres exhibit crescent-shaped artifacts caused by distortions in the surface water layer found in ambient conditions. [ABSTRACT FROM PUBLISHER]

Laser tweezers Raman spectroscopy (LTRS) has been used for the rapid detection of recombinant somatolactin protein produced in single Escherichia coli bacteria and Pichia pastoris yeast cell in the current study. A cDNA sequence encoding mature peptide of zebrafish somatolactin β was inserted into two different expression vectors and transfected into E. coli or P. pastoris yeast cells. We measured Raman spectra of single E. coli cells at different culture times following the induction with isopropyl -D-1-thiogalactopyranoside, from which the amount of the generated somatolactin proteins was obtained by the projection of the entire cell's spectrum onto the spectrum of the pure somatolactin proteins or the dot product between these two spectral vectors. We found that the intensity of the somatolactin protein-associated spectra from single E. coli cells increased as the function of the culture time, which correlates with the accumulation of recombinant proteins inside the cells. This spectral observation was supported by evidence obtained by conventional methods of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting analyses. The increased intensities of recombinant protein-associated Raman bands were also observed in another expression system, P. pastoris yeast cells. These findings demonstrate that the LTRS is a useful method for rapid sensing of recombination production in single host microorganism in vivo. [ABSTRACT FROM AUTHOR]

High-density samples of fluorescent quantum dots (QDs) were imaged using an apertureless near-field optical microscopy technique. QD fluorescence was modulated by oscillating a silicon atomic force microscope tip above an illuminated sample and a lock-in amplifier was used to suppress background from the excitation laser. Spatial resolution near 10 nm and a peak signal-to-noise ratio (SNR) of ∼60 were achieved. Individual QDs within high-density ensembles were still easily resolved (SNR>5) at a density of 14 QDs/μm2. These results have favorable implications for the eventual nanoscale imaging of viable biological systems, such as cellular membranes. [ABSTRACT FROM AUTHOR]

Biochemical studies in the human malaria parasite, Plasmodium falciparum, indicated that in addition to the pathway for synthesis of phosphatidylcholine from choline (CDP-choline pathway), the parasite synthesizes this major membrane phospholipid via an alternative pathway named the serine-decarboxylase- phosphoethanolamine-methyltransferase (SDPM) pathway using host serine and ethanolamine as precursors. However, the role the transmethylation of phosphatidylethanolamine plays in the biosynthesis of phosphatidylcholine and the importance of theSDPMpathway in the parasite's growth and survival remain unknown. Here, we provide genetic evidence that knock-out of the PfPMT gene encoding the phosphoethanolamine methyltransferase enzyme completely abrogates the biosynthesis of phosphatidylcholine via the SDPM pathway. Lipid analysis in knock-out parasites revealed that unlike in mammalian and yeast cells, methylation of phosphatidylethanolamine to phosphatidylcholine does not occur in P. falciparum, thus making the SDPM and CDP-choline pathways the only routes for phosphatidylcholine biosynthesis in this organism. Interestingly, loss of PfPMT resulted in significant defects in parasite growth, multiplication, and viability, suggesting that this gene plays an important role in the pathogenesis of intraerythrocytic Plasmodium parasites. [ABSTRACT FROM AUTHOR]

This paper is concerned mainly with the influence of the G/M ratio in the spray forming process on the size of the primary Si phase in hypereutectic Al–Si alloy. Before the hot extrusion process the growth of primary Si phase in as-deposited Al–Si alloy was observed with the increasing of the preheating temperature. The microstructure of the different samples which were extruded under the same temperature but at different extrusion ratio were compared, and the effects of the hot extrusion ratio on the refining of the primary Si phase which is distributed in the matrix of hypereutectic Al–Si alloy were discussed. According to the results of the present research the best process parameters are put forward. [Copyright &y& Elsevier]