W. Seiffert

Über Romanowsky-Farbstoffe und den Romanowsky-Giemsa-Effekt

SummaryThe Romanowsky-Giemsa staining (RG staining) has been studied by means of microspectrophotometry using various staining conditions. As cell material we employed in our model experiments mouse fibroblasts, LM cells. They show a distinct Romanowsky-Giemsa staining pattern. The RG staining was performed with the chemical pure dye stuffs azure B and eosin Y. In addition we stained the cells separately with azure B or eosin Y. Staining parameters were pH value, dye concentration, staining time etc. Besides normal LM cells we also studied cells after RNA or DNA digestion. The spectra of the various cell species were measured with a self constructed microspectrophotometer by photon counting technique. The optical ray pass and the diagramm of electronics are briefly discussed.The nucleus of RG stained LM cells, pH≃7, is purple, the cytoplasm blue. After DNA or RNA digestion the purple respectively blue coloration in the nucleus or the cytoplasm completely disappeares. Therefore DNA and RNA are the preferentially stained biological substrates.In the spectrum of RG stained nuclei, pH≃7, three absorption bands are distinguishable: They are A1 (15400 cm−1, 649 nm), A2 (16800 cm−1, 595 nm) the absorption bands of DNA-bound monomers and dimers of azure B and RB (18100 cm−1, 552 nm) the distinct intense Romanowsky band. Our extensive experimental material shows clearly that RB is produced by a complex of DNA, higher polymers of azure B (degree of assoziation p>2) and eosin Y. The complex is primarily held together by electrostatic interaction: Binding of polymer azure B cations to the polyanion DNA generates positively charged binding sites in the DNA-azure B complex which are subsequently occupied by eosin Y anions. It can be spectroscopically shown that the electronic states of the azure B polymers and the attached eosin Y interact. By this interaction the absorption of eosin Y is red shifted and of the azure B polymers blue shifted. The absorption bands of both moleculare species overlap and generate the Romanowsky band. Its strong maximum at 18100 cm−1 is due to the cosin Y part of the DNA-azure B-eosin Y complex. The discussed red shift of the eosin Y absorption is the main reason for the purple coloration of RG stained nuclei.Using a special technique it was possible to prepare an artificial DNA-azure B-eosin Y complex with calf thymus DNA as a model nucleic acid and the two dye stuffs azure B and eosin Y. Its absorption spectrum is identical with the spectrum of Romanowsky stained nuclei. This experiment demonstrates that the whole Romanowsky-Giemsa staining pattern of the nucleus is primarily produced by DNA, azure B and eosin Y.The spectrum of RG stained cytoplasm, pH≃7, consists of three absorption bands A1′ (15300 cm−1, 654 nm), A2′ (16900 cm−1, 592 nm), A3′ (17600 cm−1, 568 nm). They are attributed to monomers, dimers and polymers of azure B bound to RNA. In general A2′ and A3′ overlap strongly and generate a broad absorption band A′ (∼17000 cm−1, 588 nm). Some experimental results can be interpreted in terms of a RNA-azure B-eosin Y complex in the cytoplasm. But its concentration must be very small.Variations in the biological materials and the experimental staining conditions may alter the position and the intensity of the absorption bands but does not change the underlying molecular concept of the Romanowsky-Giemsa effect.The Romanowsky-Giemsa staining (RG staining) has been studied by means of microspectrophotometry using various staining conditions. As cell material we employed in our model experiments mouse fibroblasts, LM cells. They show a distinct Romanowsky-Giemsa staining pattern. The RG staining was performed with the chemical pure dye stuffs azure B and eosin Y. In addition we stained the cells separately with azure B or eosin Y. Staining parameters were pH value, dye concentration, staining time etc. Besides normal LM cells we also studied cells after RNA or DNA digestion. The spectra of the various cell species were measured with a self constructed microspectrophotometer by photon counting technique. The optical ray pass and the diagramm of electronics are briefly discussed. The nucleus of RG stained LM cells, pH congruent to 7, is purple, the cytoplasm blue. After DNA or RNA digestion the purple respectively blue coloration in the nucleus or the cytoplasm completely disappeares. Therefore DNA and RNA are the preferentially stained biological substrates. In the spectrum of RG stained nuclei, pH congruent to 7, three absorption bands are distinguishable: They are A1 (15400 cm-1, 649 nm), A2 (16800 cm-1, 595 nm) the absorption bands of DNA-bound monomers and dimers of azure B and RB (18100 cm-1, 552 nm) the distinct intense Romanowsky band. Our extensive experimental material shows clearly that RB is produced by a complex of DNA, higher polymers of azure B (degree of association p greater than 2) and eosin Y. The complex is primarily held together by electrostatic interaction: inding of polymer azure B cations to the polyanion DNA generates positively charged binding sites in the DNA-azure B complex which are subsequently occupied by eosin Y anions. It can be spectroscopically shown that the electronic states of the azure B polymers and the attached eosin Y interact. By this interaction the absorption of eosin Y is red shifted and of the azure B polymers blue shifted. The absorption bands of both molecular species overlap and generate the Romanowsky band. Its strong maximum at 18100 cm-1 is due to the eosin Y part of the DNA-azure B-eosin Y complex. The discussed red shift of the eosin Y absorption is the main reason for the purple coloration of RG stained nuclei. Using a special technique it was possible to prepare an artificial DNA-azure B-eosin Y complex with calf thymus DNA as a model nucleic acid and the two dye stuffs azure B and eosin Y.(ABSTRACT TRUNCATED AT 400 WORDS)

TIME-RESOLVED MICROFLUOROMETRIC STUDY OF THE BINDING SITES OF LIPOPHILIC CATIONIC PYRENE PROBES IN MITOCHONDRIA OF LIVING HELA CELLS

Lipophilic dye cations specifically bind to the mitochondria of living cells. Using fluorescent dyes, the mitochondria can easily be observed with a fluorescence microscope. Electron microscopy has shown that the dyes are bound to the inner mitochondrial membranes and the cristae. Using time-resolved fluorescence microscopy we have investigated, whether the dye molecules are preferentially accumulated at the strongly hydrophobic protein complexes of energy metabolism or at the lipids of the inner membrane system. In order to use our nanosecond-pulsed laser fluorometer we synthesized specially designed lipophilic pyrene cations with S1 lifetimes in the nanosecond domain, which specifically stain mitochondria in living HeLa cells. Model experiments with artificial membranes such as liposomes, proteoliposomes and also protein complexes have shown that the fluorescence is strongly quenched by oxygen if the pyrene probes are bound to lipids. Binding to proteins causes a much smaller quenching effect. In artificial systems, all decays were single exponential. This is in contrast with incubated HeLa cells, which showed double-exponential fluorescence decays. Comparing these with the artificial systems we came to the conclusion that in HeLa cells the long-lived species 1 are pyrene probes preferentially bound to the proteins of the inner mitochondrial membranes. The short-lived species 2 is caused by fluorescence resonance energy transfer from the pyrene probes as donors to cytochromes of the inner membranes as acceptors. From our decay data we estimated a mean distance between donor and acceptor of about 40 A. This is the same order of magnitude as the mean diameters of several mitochondrial protein complexes. Therefore we assumed that species 2 are pyrene probes bound either to mitochondrial proteins with cytochromes as constituents or to the interface between these proteins and the phospholipids of the membranes. Thus both species 1 and species 2 are spatially related to mitochrondrial proteins. This agrees with the observation that respiration of HeLa cells as well as cytochrome c oxidase vesicles (COVs) are inhibited with increasing concentration of pyrene probes. Finally, we studied the photodynamic effect on irradiation of HeLa cells and of COVs after incubation with lipophilic pyrene and porphyrine cations.

Spektroskopische und thermodynamische Untersuchungen zur Bindung von Azur B an Chondroitinsulfat und zur Bindungsgeometrie des metachromatischen Farbstoffkomplexes

The binding of azur B to chondroitin sulfate (CHS) was investigated using absorption spectroscopy. In aqueous solutions it is possible to distinguish three different dye species with absorption bands at 646, 597, and 555 nm. They are assigned to monomers, dimers, and higher aggregates of azure B, which become bound to CHS as the dye concentration (CD) increases. The short-wavelength band (555 nm) causes metachromasia in stained histological materials. When saturation occurs, the metachromatic azure B-CHS complex has a 1:1 composition, i.e., each anionic SO-4 and COO(-)-binding site of CHS binds one dye cation. The composition of the saturated metachromatic complex was determined by spectrophotometric and conductometric titration of CHS with azure B, while the SO-4 and COO- content of CHS was determined by conductometric titration of CHS-acid with NaOH. The binding isotherm of azure B to CHS was determined using gelpermeation chromatography. The isotherm can be described by the model of cooperative binding of ligands to linear biopolymers. We found good agreement between theoretical predictions and experimental findings in the range of 0 less than r less than 0.8 (r = the fraction of occupied binding sites). Using a Schwarz plot, we determined the binding constants of nucleation (Kn = 2.5 X 10(3) M-1) and aggregation (Kq = 1.2 X 10(5) M-1), as well as the cooperativity parameter (q = 50), T = 295 K. With increasing CD, the strong cooperativity of the dye binding favors the formation of metachromatic aggregates rather than monomers and dimers. From the temperature dependence of Kq we evaluated the standard binding enthalpy (delta Hoq = -20.0 kJ mol-1) and entropy (delta Soq = 29.7 JK-1 mol-1) of the cooperative dye binding. The binding was found to be strongly exothermic and accompanied by a thermodynamically favorable entropy increase, this being typical of hydrophobic interactions. Solid azure B-CHS complexes were prepared according to a special dialytic technique and were studied using a microspectrophotometer equipped with a polarizer and an analyzer. The metachromatic 1:1 complex has a broad, intense absorption band whose main peak occurs at 560 nm. This corresponds with the maximum of the metachromatic dye complex in aqueous solution, i.e. 555 nm. The CHS chains of the azure B-CHS complex can be mechanically aligned in a preferred direction (k). We were able to prepare excellently orientated and very fine dye-CHS films which were birefringent and dichroic - the more birefringent, the better the mechanical orientation.(ABSTRACT TRUNCATED AT 400 WORDS)SummaryThe binding of azur B to chondroitin sulfate (CHS) was investigated using absorption spectroscopy. In aqueous solutions it is possible to distinguish three different dye species with absorption bands at 646, 597, and 555 nm. They are assigned to monomers, dimers, and higher aggregates of azure B, which become bound to CHS as the dye concentration (CD) increases. The short-wavelength band (555 nm) causes metachromasia in stained histological materials. When saturation occurs, the metachromatic azure B-CHS complex has a 1:1 composition, i.e., each anionic SO−4-and COO−-binding site of CHS binds one dye cation. The composition of the saturated metachromatic complex was determined by spectrophotometric and conductometric fitration of CHS with azure B, while the SO−4and COO− content of CHS was determined by conductometric titration of CHS-acid with NaOH.The binding isotherm of azure B to CHS was determined using gelpermeation chromatography. The isotherm can be described by the model of cooperative binding of ligands to linear biopolymers. We found good agreement between theoretical predictions and experimental findings in the range of 0<r<0.8 (r=the fraction of occupied binding sites). Using a Schwarz plot, we determined the binding constants of nucleation (Kn=2.5·103 M−1) and aggregation (Kq=1.2·105 M−1), as well as the cooperativity parameter (q=50), T=295 K. With increasing CD, the strong cooperativity of the dye binding favors the formation of metachromatic aggregates rather than monomers and dimers. From the temperature dependence of Kq we evaluated the standard binding enthalpy (†Hqo=20.0 kJ mol−1) and entropy (†Sqo=29.7 JK−1 mol−1) of the cooperative dye binding. The binding was found to be strongly exothermic and accompanied by a thermodynamically favorable entropy increase, this being typical of hydrophobic interactions.Solid azure B-CHS complexes were prepared according to a special dialytic technique and were studied using a microspectrophotometer equipped with a polarizer and an analyzer. The metachromatic 1:1 complex has a broad, intense absorption band whose main peak occurs at 560 nm. This corresponds with the maximum of the metachromatic dye complex in aqueous solution, i.e. 555 nm. The CHS chains of the azure B-CHS complex can be mechanically aligned in a preferred direction (k). We were able to prepare excellently orientated and very fine dye-CHS films which were birefringent and dichroic-the more birefringent, the better the mechanical orientation. The site of best orientation within the preparations was selected according to the quality of the birefringence, and this region was then measured with a microspectrophotometer using linearly polarized light. By setting the polarizer (ep) parallel and perpendicular to k, we obtained the dichroic ratio (d) of the light absorbance (E, d=E∥/E⊥). A dichroic ratio of d=0.1 was determined for the 560 nm absorption band of the metachromatic dye complex. Therefore, the transition moment (m) of the dye molecules is polarized almost perpendicularly to the preferred direction, k, m⊥k. The moment (m) of the

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Über eine Bertalanffy-analoge Fluorochromierung mit 3-Dimethylamino-6-methoxyacridin

transition of the azure B chromophore lies in the molecular plane in the direction of the long molecular axis. For this reason, the long axes of the dye molecules and the longitudinal CHS axis of the metachromatic complex are approximately perpendicular to each other. Adjacent dye molecules are in van der Waals contact, and the π orbitals interact. This interaction causes the changes in the absorption spectra upon the formation of the complex. CHS has a helical conformation, so that the metachromatic complex looks something like a winding stairway, the central pillar being the CHS helix and the steps being the dye molecules.Sagittal sections of bovine cornea were stained with azure B, and the absorption spectra were measured microphotometrically using polarized light. The substantia propria corneae consists of morphologically well-orientated fibers made of a CHS-containing proteoglycan. We found the main peak of absorption to be at 565 nm, indicating metachromatically bound dye molecules. Its dichroic ratio of d=0.26 (k=the direction of the fibers) showed that the long axes of the dye molecules are orientated perpendicularly to the fibers of the substantia propria.

Modelluntersuchungen zur Struktur des purpurnen Farbstoffkomplexes der Giemsa-Färbung

SummaryA reproducible Romanowsky-Giemsa staining (RGS) can be carried out with standardized staining solutions containing the two dyes azure B (AB) and eosin Y (EY). After staining, cell nuclei have a purple coloration generated by DNA-AB-EY complexes. The microspectra of cell nuclei have a sharp and intense absorption band at 18 100 cm−1 (552 nm), the so called Romanowsky band (RB), which is due to the EY chromophore of the dye complexes. Other absorption bands can be assigned to the DNA-bound AB cations.Artificial DNA-AB-EY complexes can be prepared outside the cell by subsequent staining of DNA with AB and EY. In the first step of our staining experiments we prepared thin films of blue DNA-AB complexes on microslides with 1:1 composition: each anionic phosphodiester residue of the nucleic acid was occupied by one AB cation. Microspectrophotometric investigations of the dye preparations demonstrated that, besides monomers and dimers, mainly higher AB aggregates are bound to DNA by electrostatic and hydrophobic interactions. These DNA-AB complexes are insoluble in water. Therefore it was possible to stain the DNA-AB films with aqueous EY solutions and also to prepare insoluble DNA-AB-EY films in the second step of the staining experiments. After the reaction with EY, thin sites within the dye preparations were purple. The microspectra of the purple spots show a strong Romanowsky band at 18 100 cm−1. Using a special technique it was possible to estimate the composition of the purple dye complexes. The ratio of the two dyes was approximately EY:AB≈1:3. The EY anions are mainly bound by hydrophobic interaction to the AB framework of the electrical neutral DNA-AB complexes. The EY absorption is red shifted by the interaction of EY with the AB framework of DNA-AB-EY. We suppose that this red shift is caused by a dielectric polarization of the bound EY dianions.The DNA chains in the DNA-AB complexes can mechanically be aligned in a preferred direction k. Highly orientated dye complexes prepared on microslides were birefringent and dichroic. The orientation is maintained during subsequent staining with aqueous EY solutions. In this way we also prepared highly orientated purple DNA-AB-EY complexes on microslides. The light absorption of both types of dye complexes was studied by means of a microspectrophotometer equipped with a polarizer and an analyser. The sites of best orientation within the dye preparations were selected under crossed nicols according to the quality of birefringence. Subsequently, the absorption spectra of the highly orientated dye complexes were measured with plane polarized light. We found that the transition moments, mAB, of the bound AB cations in DNA-AB and DNA-AB-EY are orientated almost perpendicular to k, i.e. mAB⊥k. On the contrary, the transition moments, mEY, of the bound EY anions in DNA-AB-EY are polarized parallel to k, i.e. mEY ∥ k. The transition moments mAB and mEY lay in the direction of the long axes of the AB and EY chromophores. For that reason, in both DNA-AB and DNA-AB-EY the long molecular axes of the AB cations are orientated approximately perpendicular to the DNA chains, while the long molecular axes of the EY chromophores are polarized in the direction of the DNA chains. Therefore, in DNA-AB-EY the long axes of AB and EY are perpendicular to each other, mAB⊥mEY. This molecular arrangement fully agrees with our quantitative measurements and with the theory of the absorption of plane polarized light by orientated dye complexes, which has been developed and discussed in detail.

NMR- und UV-spektroskopische untersuchungen zur struktur des photoprodukts von acridin-n-oxyd

Azure B is the most important Romanowsky dye. In combination with eosin Y it produces the well known Romanowsky-Giemsa staining pattern on the cell. Usually commercial azure B is strongly contaminated. We prepared a sample of azure B-BF4 which was analytically pure and had no coloured impurities. The substance was used to redetermine the molar extinction coefficient epsilon (v)M of monomeric azur B in alcoholic solution. In the maximum of the long wavelength absorption at v = 15.61 kK (lambda = 641 nm) the absorptivity is epsilon (15.61)M = (9.40 +/- 0.15) x 10(4)M-1 cm-1. This extinction coefficient may be used for standardization of dye samples. In aqeuous solution azur B forms dimers and even higher polymers with increasing concentration. The dissociation constant of the dimers, K = 2,2 x 10(-4)M (293 K), and the absorption spectra of pure monomers and dimers in water have been calculated from the concentration dependence of the spectra using an iterative procedure. The molar extinction coefficient of the monomers at 15.47 kK (646 nm) is epsilon (15.47)M = 7.4 x 10(4)M-1 cm-1. The dimers have two long wavelength absorption bands at 14.60 and 16.80 kK (685 and 595 nm) with very different intensities 2 x 10(4) and 13.5 x 10(4)M-1 cm-1. The spectrum of the dimers in aqueous solution is in agreement with theoretical considerations of Förster (1946) and Levinson et al. (1957). It agrees with an antiparallel orientation of the molecules in the dimers. It may be that dimers bound to a substrate in the cell have another geometry than dimers in solution. In this case the weak long wavelength absorption of the dimers can increase.SummaryAzure B is the most important Romanowsky dye. In combination with eosin Y it produces the well known Romanowsky-Giemsa staining pattern on the cell. Usually commercial azure B is strongly contaminated. We prepared a sample of azure B-BF4 which was analytically pure and had no coloured impurities. The substance was used to redetermine the molar extinction coefficient