A sensitive confocal Raman microspectrometer was employed to record spectra of nuclei and cytoplasmic regions of single living human granulocytes. Conditions were used that ensured cell viability and reproducibility of the spectra. Identical spectra were obtained from the nuclei of neutrophilic, eosinophilic, and basophilic granulocytes, which yield information about DNA and protein secondary structure and DNA-protein ratio. The cytoplasmic Raman spectra of the three cell types are very different. This was found to be mainly due to the abundant presence of peroxidases in the cytoplasmic granules of neutrophilic granulocytes (myeloperoxidase) and eosinophilic granulocytes (eosinophil peroxidase). Strong signal contributions of the active site heme group(s) of these enzymes were found. This paper illustrates the potentials and limitations for Raman spectroscopic analysis of cellular constituents and processes.

Real-time single-molecule fluorescence detection using confocal and near-field scanning optical microscopy has been applied to elucidate the nature of the ''on-off'' blinking observed in the Ser-65 3 Thr (S65T) mutant of the green fluorescent protein (GFP). Fluorescence time traces as a function of the excitation intensity, with a time resolution of 100 s and observation times up to 65 s, reveal the existence of a nonemissive state responsible for the long dark intervals in the GFP. We find that excitation intensity has a dramatic effect on the blinking. Whereas the time during which the fluorescence is on becomes shorter as the intensity is increased, the off-times are independent of excitation intensity. Statistical analysis of the on-and off-times renders a characteristic off-time of 1.6 ؎ 0.2 s and allows us to calculate a transition yield of Ϸ0.5 ؋ 10 ؊5 from the emissive to the nonemissive state. The saturation excitation intensity at which on-and off-times are equal is Ϸ1.5 kW͞cm 2 . On the basis of the single-molecule data we calculate an absorption cross section of 6.5 ؋ 10 ؊17 cm 2 for the S65T mutant. These results have important implications for the use of the GFP to follow dynamic processes in time at the single-molecular level. Since the cloning and subsequent expression of the green fluorescent protein (GFP) from the jellyfish Aequorea victoria (1, 2) the research interest for this protein has increased dramatically. The protein has been used successfully in a large and ever-growing number of applications, including gene expression and cell dynamics (3, 4). It is the only cloned protein that exhibits strong intrinsic fluorescence without the need of external chromophores. In the native protein [wild-type (wt)-GFP] the chromophore is formed in an autocatalytic, posttranslational cyclization and oxidation of the tripeptide unit at residues 65-67 (2, 5, 6). The wt-GFP absorbs blue light at Ϸ395 nm, with a weak peak at Ϸ475 nm, and emits green light at Ϸ508 nm (7). Substitution of one or more amino acids at or in close proximity to the chromophore results in mutants with different absorption and emission properties, and in some cases, improved emission and photostability (5,8,9). A widely used mutant is the S65T, in which Ser-65 is replaced by Thr (10). The mutant shows only an absorption peak at Ϸ475 nm, has larger absorption cross section than wt-GFP, and shows no photo-isomerization (8, 9).Because of the rapidly increasing number of applications, great attention has been focused on the photophysical properties of the wt-GFP and a number of its mutants. Investigation of the photophysical properties has been carried out in ensemble measurements, at room and low temperatures (7,(11)(12)(13)(14), and at the single-molecular level (15)(16)(17)(18)(19). When molecules are observed individually, the fluorescence emission of GFP shows intensity fluctuations, on-off blinking, and fluorescence switching, a behavior that is hidden in ensemble experiments. These intriguing phenomena also manifest in many other...

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 C 2′ -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 helix.The secondary structure of poly(dG-dC)‚poly(dG-dC) in aqueous solutions of low ionic strength and at physiological pH is generally recognized as a double-stranded helix with the strands connected by Watson-Crick base pairing, the so-called B-form. In a high-salt environment, it was established, by means of CD and Raman spectra, that the polynucleotide has a left-handed double-helical structure (Pohl & Jovin, 1972;Thamann et al., 1981;Benevides & Thomas, 1983).Apart from studies on effects of salt concentration, several investigations have been performed on the influence of pH on DNA conformation. Courtois et al. (1968) used optical rotatory dispersion and suggested a conformational change of guanine residues in DNA upon acid titration between pH 3 and 4. It was hypothesized as a possible interpretation of their results that upon protonation on guanine N7, guanine becomes unstacked and rotates out of the helix, reverses from the anti into the syn position, pairs in a Hoogsteen manner, and thus shares the proton with N3 on cytosine. Zimmer and Triebel (1969) concluded from spectrophotometric pHtitration measurements at 20 mM [K + ] that reversible and irreversible conformational changes occur in DNA upon acid treatment. From viscosimetric and spectrophotometric investigations on DNA at different pH values and ionic strengths, it was shown by Kas'yanenko et al. (1986) that DNA is in a double-helical form until at least pH 3.5 at 0.1 M salt concentration. Chen (1984) suggests from spectroscopic studies on the salt titration for poly(dG-dC)‚poly(dGdC) in solutions with pH 7.0 and pH 3.8 that base protonation facilitates the B to Z interconversion.The possible presence of prot...

With (resonance) Raman microscospectroscopy, it is possible to investigate the chemical constitution of a very small volume (0.5 fl) in a living cell. We have measured resonance Raman spectra in the cytoplasm of living normal, myeloperoxidase (MPO)-deficient, and cytochrome b558-deficient neutrophils and in isolated specific and azurophilic granule fractions, using an excitation wavelength of 413.1 nm. Similar experiments were performed after reduction of the redox centers by the addition of sodium dithionite. The specific and azurophilic granules in both redox states appeared to have clearly distinguishable Raman spectra when exciting at a wavelength of 413.1 nm. The azurophilic granules and the cytochrome b558-deficient neutrophils showed Raman spectra similar to that of the isolated MPO. The spectra of the specific granules and the MPO-deficient neutrophils corresponded very well to published cytochrome b558 spectra. The resonance Raman spectrum of the cytoplasmic region of normal neutrophilic granulocytes could be fitted with a combination of the spectra of the specific and azurophilic granules, which shows that the Raman signal of neutrophilic granulocytes mainly originates from MPO and cytochrome b558, at an excitation wavelength of 413.1 nm.

A confocal Raman microspectrometer was used to investigate the influence of Ca2+ cations on low pH-induced DNA structural changes. The effects of Ca2+ cations on the protonation mechanism of opening AT and changing the protonation of GC base pairs in DNA are discussed. Based on the observation that the midpoint of the transition of Watson-Crick GC base pairs to protonated GC base pairs lies at around pH 3 (analyzing the 681 cm(-1) line), measurements were carried out on calf thymus DNA at neutral pH and pH 3 in the presence of low and high concentrations of Ca2+ cations. Raman spectra show that low concentrations of Ca2+ cations partially protect DNA against protonation of cytosine (characteristic line at 1262 cm(-1)) and do not protect adenine (characteristic line at 1304 cm(-1)) and the N(7) of guanine (line at 1488 cm(-1)) against binding of H+. High Ca2+ concentrations can prevent protonation of cytosine and protonation of adenine (little disruption of AT pairs). Analyzing the line at 1488 cm(-1), which obtains most of its intensity from a guanine vibration, high salt was also found to protect the N(7) of guanine against protonation.

Low-pH-induced DNA structural changes were investigated in the pH range 6.8-2.10 by Raman microspectroscopy. Measurements were carried out on calf thymus DNA in the presence of low concentrations of Mg 2+ ions. Vibrational spectra are presented in the wavenumber region 500-1650 cm −1 . Large changes in the Raman spectra of calf-thymus DNA were observed on lowering the pH value. These are due to protonation and unstacking of the DNA bases during DNA melting and also to changes in the DNA backbone conformation. The intensities of the Raman bands of guanine (681 cm −1 /, adenine (728 cm −1 /, thymine (752 cm −1 / and cytosine (785 cm −1 /, typical of the C2 -endo-anti conformation of B-DNA, are discussed. The B-form marker near 835 cm −1 and the base vibrations in the higher wavenumber region (1200-1680 cm −1 / are analysed. Effects of low pH value upon the protonation mechanism of opening AT and changing the protonation of GC base pairs in DNA are discussed.

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The pathogenesis of Parkinson's disease that is the second most common neurodegenerative disease is associated with formation of different aggregates of α-synuclein (ASN), namely oligomers and amyloid fibrils. Current research is aimed on the design of fluorescent dyes for the detection of oligomeric aggregates, which are considered to be toxic and morbific spices. Fluorescent properties of series of benzothiazole trimethine and pentamethine cyanines were characterized in free state and in presence of monomeric, oligomeric and fibrilar ASN. The dyes with wide aromatic systems and bulky phenyl and alkyl substituents that are potentially able to interact with hydrophobic regions of oligomeric aggregates were selected for the studies. For majority of studied dyes noticeable changes in fluorescence characteristics were shown in the presence of fibrillar or oligomeric ASN, while the dyes slightly responded on the presence of monomeric protein. For pentamethine cyanine SL-631 and trimethine cyanine SH-299 certain specificity to oligomeric aggregates over fibrils was observed. Using these dyes at 10(-6) M concentration permits the detection of oligomeric ASN in the concentrations range of at least 0.2-2 microM. Pentamethine cyanine SL-631 is proposed as dye for fluorescent detection of oligomeric aggregates of ASN, while trimethine cyanine SH-299 is shown to be a sensitive probe both on oligomeric and fibrillar ASN. It is proposed that wide aromatic system of SL-631 pentamethine dye molecule could better fix on the less dense and structured oligomeric formation, while less bulky and more "crescent-shape" molecule of trimethine dye SH-299 could easier enter into the groove of beta-pleated structure.