A. Naujok

[The fluorescent staining of mitochondria in living HeLa- and LM-cells with new acridine dyes (author's transl)].

SummaryThe fluorescent staining of mitochondria in living cells with new acridine dyes is reported. The fluorescent dyes used are derivatives of acridine orange (AO) and of 3-amino-6-methoxyacridine (AMA) with various residues in 9- or 10-position (Scheme 1). They are either permanent cationic dyes or cations which are formed by protonation in the culture medium. HeLa cells and mouse fibroblasts (LM cells) have been used for our staining experiments. On favourable conditions we succeeded in staining the mitochondria not only orthochromatically but also metachromatically. Photodynamical effects which have been observed during the exposure of the stained cells in the fluorescence microscope are described. The residues in 9- or 10-position favour the dye accumulation in the mitochondria. Vital staining with the basic compounds AO and AMA however leads to the formation of metachromatically stained lysosomes in the orthochromatically stained cytoplasm. The dye 3-amino-6-methoxy-9-(2-hydroxyethyl)acridine stains the nucleus of living cells.ZusammenfassungEs wird über die Fluorochromierung von Mitochondrien in lebenden Zellen mit neuen Acridinfarbstoffen berichtet. Die verwendeten Fluorochrome sind Derivate von Acridinorange (AO) und von 3-Amino-6-methoxyacridin (AMA) mit verschiedenen Resten in 9- bzw. 10-Stellung (Formelschema 1). Sie sind entweder permanente Farbstoffkationen oder liegen im Kulturmedium als Kationen vor. HeLa-Zellen und Mäusefibroblasten (LM-Zellen) wurden fluorochromiert. Unter günstigen Bedingungen gelang es, die Mitochondrien nicht nur orthochromatisch sondern auch metachromatisch zu färben. Über photodynamische Effekte, die bei der Bestrahlung unter dem Fluoreszenzmikroskop auftreten, wird berichtet. Die Reste in 9- bzw. 10-Stellung begünstigen die Farbstoffakkumulation in den Mitochondrien. vitalfärbung mit den Grundkörpern AO bzw. AMA ergibt demgegenüber metachromatisch gefärbte Lysosomen im orthochromatisch gefärbten Cytoplasma. Der Farbstoff 3-Amino-6-methoxy-9-(2-hydroxyethyl)-acridin fluorochromiert den Kern lebender Zellen.The fluorescent staining of mitochondria in living cells with new acridine dyes is reported. The fluorescent dyes used are derivatives of acridine orange (AO) and of 3-amino-6-methoxyacridine (AMA) with various residues in 9- or 10-position (Scheme 1). They are either permanent cationic dyes or cations which are formed by protonation in the culture medium. HeLa cells and mouse fibroblasts (LM cells) have been used for our staining experiments. On favourable conditions we succeeded in staining the mitochondria not only orthochromatically but also metachromatically. Photodynamical effects which have been observed during the exposure of the stained cells in the fluorescence microscope are described. The residues in 9- or 10-position favour the dye accumulation in the mitochondria. Vital staining with the basic compounds AO and AMA however leads to the formation of metachromatically stained lysosomes in the orthochromatically stained cytoplasm. The dye 3-amino-6-methoxy-9-(2-hydroxyethyl)acridine stains the nucleus of living cells.

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)

[Hydrophobic acridine dyes for fluorescence staining of mitochondria in living cells. 2. Comparison of staining of living and fixed Hela-cells with NAO and DPPAO].

The hydrophobic fluorescence dyes NAO and DPPAO (see scheme of structural formulae) stain the mitochondria of living HeLa-cells. The trans-membrane potential favours the dye accumulation of the cation NAO and supports the hydrophobic interaction of the dye with the mitochondrial membrane lipids and proteins. The lecithin-like dye DPPAO is electrical neutral. Its binding to mitochondria of living cells is only caused by hydrophobic interaction. NAO and DPPAO stain also the mitochondria of glutaraldehyde fixed HeLa-cells in aqueous medium. Fluorescence staining occurs even after extraction of the lipids of the cell with acetone. We suppose that the dye accumulation in the mitochondria of the fixed cells is caused by the hydrophobic interaction between the dyes and the very hydrophobic mitochondrial lipids and proteins.SummaryThe hydrophobic fluorescence dyes NAO and DPPAO (see scheme of structural formulae) stain the mitochondria of living HeLa-cells. The trans-membrane potential favours the dye accumulation of the cation NAO and supports the hydrophobic interaction of the dye with the mitochondrial membrane lipids and proteins. The lecithinlike dye DPPAO is electrical neutral. Its binding to mitochondria of living cells is only caused by hydrophobic interaction. NAO and DPPAO stain also the mitochondria of glutaraldehyde fixed HeLa-cells in aqueous medium. Fluorescence staining occures even after extraction of the lipids of the cell with acetone. We suppose that the dye accumulation in the mitochondria of the fixed cells is caused by the hydrophobic interaction between the dyes and the very hydrophobic mitochondrial lipids and proteins.

Effects of ethidium bromide, tetramethylethidium bromide and betaine B on the ultrastructure of HeLa cell mitochondria in situ

SummarySeveral investigators have described the ultrastructural changes that occur in the mitochondria of cells in tissue cultures after treatment with the drug ethidium bromide (E). The mitochondria swell and the cristae become greatly altered and finally disappear; in the cristae-free region of the matrix electron-dense granules can be observed. It has been assumed that intercalation of E between the base pairs of the mitochondrial DNA induces the formation of the granular inclusions. To investigate whether intercalation is really the initial step in the generation of dense granules inside the matrix, we performed a comparative incubation study of HeLa-cell mitochondria in situ using three closely related dyes (D), i.e. E, tetramethylethidium bromide (TME) and betaine B (B). They strongly differ with regard to their affinity for DNA and their ability to cross membranes. E was used as a reference dye. TME does not intercalate, but is externally bound to DNA only weakly. The neutral B is not bound at all, but can cross membranes more easily than the cation E. Moreover, in aqueous solutions at pH≃7.0, B is in equilibrium with its protonated cation BH. BH and E have almost equal affinities for DNA. Therefore B may quickly pass the inner mitochondrial membranes and the cristae, and should then be bound inside the matrix, thus forming a BH-DNA complex. On the assumption that intercalation is necessary for the generation of intramitochondrial electron-dense bodies, we predicted that BH/B should be more efficient than E, while TME should be relatively ineffective. In experiments using HeLa cells, these predictions were found to be inaccurate. E, TME and BH/B produced almost the same mitochondrial alterations, but at different concentrations and after different incubation periods. In contrast to our expectations TME was much more effective than E and BH/B, with the last two behaving rather similarly.Therefore, it seems unlikely that the drugs penetrate the inner mitochondrial membrane system by simple physical diffusion or that intercalation is the preliminary step for the generation of dense granules inside the matrix. Instead, we assume that hydrophobic interaction between the dye cations E, BH and TME and the cristae is the main cause of the mitochondrial changes. The favoured binding partner of the dye cations may be the divalent anion, cardiolipin: this phospholipid is an essential part of the inner membrane system but is absent in other membranes of cells. By distributing the dyes between a lipophilic phase and water, it was shown that TME is more lipophilic than E and BH; this may explain the greater effectiveness of TME. The bound dye cations disturb the organization of the cristae, which become altered and finally disappear. We assume that the electron-dense granules in the matrix are mainly composed of the dyes and former membrane materials such as phospholipids and proteins, as well as perhaps some other hydrophobic matrix materials. This would also explain why it was impossible to digest the dense granules by DNase treatment. The drugs enter the mitochondrial matrix by disordering and finally destroying the cristae.

The concentration jump method

Lipophilic cationic fluorescent dyes (D) specifically stain the mitochondria of living cells. A perfusion chamber for cell cultures is described, which can be used to determine the kinetics of vital staining of the mitochondria of single selected cells in situ. In these experiments styrylpyridinium dyes and cultures of HeLa cells were used. The dyes differ strongly in their lipophilic properties; Rm values and the partition coefficients Po/w between n-octanol (o) and water (w) were determined in order to characterize their lipophilicity. In the thermostat-regulated chamber the concentration of the dye CD can be increased from CD=0 to CD>0 within a few seconds (concentration jump). Thus, the time t=0 for the beginning of the vital staining and the dye concentration in the cell medium during the staining experiment, CD=const., are unambiguously defined. The concentration of the dye, Cb, which is bound to the mitochondria (b), is proportional to the intensity of the fluorescence Ib. On the other hand, the free dye molecules (f) in the aqueous medium exhibit practically no fluorescence, If≪Ib. The intensity of the fluorescence I=Ib was measured as a function of time t; the measured values were corrected for photobleaching. The fluorescence intensity I(t) at first increases linearly with t and reaches a saturation value for t → ∞. In the linear range of I(t) the flow Jo=(dI/dt)o of the dye into the cell depends strongly on the dye concentration and increases linearly with CD. The concentration range CD=10−9−10−5M at 37° C was investigated. From the linear correlation between Jo and CD it follows that the kinetics of the vital staining of mitochondria is controlled by diffusion. At t=0 the flow of the xenobiotic agent through the cell membrane determines the rate of staining. The slope dJo/dCD of the plot Jo vs CD describes the efficiency of dye accumulation at the mitochondria and strongly increases with increasing lipophilicity of the dye molecules. Thus lipophilic dyes pass through the cell membrane more easily than less lipophilic molecules.

Vitalfluorochromierung von Chromosomen und Kernstrukturen in LM- und HeLa-Zellen mit neuen Acridinfarbstoffen

SummaryFluorescent staining of chromosomes and nuclear structures (nucleolus associated chromatin) in living HeLa- and LM-cells (mouse fibroblasts) with new acridine dyes is reported. The dyes have aminoethylgroups in 9-position with different endgroups at this residue (scheme of structures). Dyes without these 9-substituents only induce the formation of lysosomes. An exceptional position on vital staining of chromosomes and nuclear chromatin has the dye 3-amino-6-methoxy-9-(2-hydroxyethylamino)acridine 1. Concentrations of 10−3 M can be used in vital staining experiments. Measuring the consumption of oxygen we could demonstrate that the dye has no effect on the activity of respiration even at these high dye concentrations. Therefore we conclude that we have really observed vital staining and not postvital staining of chromosomes and nuclear chromatin. Similar properties has the well known vital dye acridine orange.ZusammenfassungEs wird über die Fluorochromierung von Chromosomen und von Kernstrukturen (Nucleolus-assoziiertes Chromatin) in lebenden HeLa- und LM-Zellen (Mäusefibroblasten) mit neuen Acridinfarbstoffen berichtet. Die Farbstoffe haben in 9-Stellung des Acridingerüsts eine Aminoethylgruppe mit verschiedenen Endgruppen (Formelschema). Fehlt der 9-Substituent, so werden bei Einwirkung des Farbstoffs nur Lysosomen gebildet. Besonders bewährt hat sich bei der Vitalfluorochromierung von Chromosomen und Kernchromatin der Farbstoff 3-Amino-6-methoxy-9-(2-hydroxyetylamino)acridin 1. Konzentrationen von 10−3 M können bei der Vitalfärbung angewendet werden. Durch Messung des Sauerstoffumsatzes konnte gezeigt werden, daß die Atmungsaktivität der Zellen selbst bei diesen hohen Konzentrationen durch den Farbstoff nicht beeinflußt wird. Daraus schließen wir, daß wir tatsächlich Vitalfärbungen und nicht Postvitalfärbungen beobachtet haben. Ähnliche Eigenschaften hat der bekannte Vitalfarbstoff Acridinorange.Fluorescent staining of chromosomes and nuclear structures (nucleolus associated chromatin) in living HeLa- and LM-cells (mouse fibroblasts) with new acridine dyes is reported. The dyes have aminoethylgroups in 9-position with different end groups at this residue (scheme of structures). Dyes without these 9-substituents only induce the formation of lysosomes. An exceptional position on vital staining of chromosomes and nuclear chromatin has the dye 3-amino-6-methoxy-9-(2-hydroxyethylamino)acridine 1. Concentrations of 10(-3) M can be used in vital staining experiments. Measuring the consumption of oxygen we could demonstrate that the dye has no effect on the activity of respiration even at these high dye concentrations. Therefore we conclude that we have really observed vital staining and not postvital staining of chromosomes and nuclear chromatin. Similar properties has the well known vital dye acridine orange.

Influence of trans-membrane potential and of hydrophobic interactions on dye accumulation in mitochondria of living cells

The lipophilic cationic fluorescent dye azopentylmethylindocarbocyanine (APMC) specifically stains the mitochondria in living cells. The dye contains a photosensitive diazirine ring and is suitable for photoaffinity labelling of mitochondrial proteins. By a combination of photoaffinity labelling of cell cultures of mouse fibroblasts (LM) with APMC, lysis of the labelled cells, subsequent micro-gel electrophoresis and detection of the fluorescence of the labelled proteins in the gel lanes with a sensitive microfluorimeter, we determined the number, apparent molecular masses, and relative intensity of the labelled proteins. In LM cells, three proteins with apparent molecular masses of 31, 40, and 74 kDa were labelled with high intensity, and proteins of 28, 29, 44, 48, 49, 66, and 105 kDa with low intensity. Two effects mainly determine the binding of lipophilic dye cations to mitochondrial proteins in living cells: (1) interaction of the trans-membrane potential of the inner mitochondrial membrane with the dye cations; and (2) hydrophobic interactions between the strongly lipophilic proteins of the inner membrane and the lipophilic dye molecules. Preincubation of the cell cultures with drugs that dissipate the trans-membrane potential, such as valinomycin, 2,4-dinitrophenol (DNP) and 3-chlorcarbonylcyanidephenylhydrazon (CCCP), strongly reduces or even prevents APMC labelling of mitochondrial proteins. The influence of hydrophobic interactions was investigated by competitive staining experiments using dyes with very different lipophilic properties. The lipophilicity of the dyes was characterized by their Rm values in reversed phase thin-layer chromatography. Prestaining with an excess of strongly lipophilic cationic acridine and phenanthridine dyes, such as pentyl acridinium orange chloride (PAO), nonyl acridinium orange chloride (NAO) and tetramethylpropidium chloride (MP), respectively, completely prevents protein labelling with APMC. Obviously, the dyes occupy the same mitochondrial binding sites as APMC. At equal concentrations the intensity of the 40-kDa signal is strongly reduced, whereas the 31-kDa and 74-kDa signals are unaffected. Using phenanthridine dyes with lower lipophilicity, namely propidium chloride (P), M, and N reduces the peak of the 40-kDa protein in APMC labelling, indicating that the 40-kDa protein preferentially binds lipophilic dye cations.

Über die Vitalfluorochromierung des Kernchromatins und der Chromosomen von HeLa-Zellen mit AMHA und die Bindung des Acridinfarbstoffs an DNA

SummaryThe fluorochrome AMHA (3-amino-6-methoxy-9-(2-hydroxyethylamino)acridine) stains the nuclear chromatin and the chromosomes of living HeLa cells. At relatively low dye concentrations CF≤10−4M and short incubation periods tI≤2 h cell growth is not affected by the drug. But at higher CF and longer tI the population doubling time of the cell cultures rapidly increases, and finally the cells die.In vital staining experiments the dye AMHA preferentially binds to the DNA of the nuclei and to the chromosomes of the cells, respectively. The dye binding to DNA has been proved by the absorption and emission microspectra of the stained cells, and by the comparison with authentic spectra of AMHA bound to DNA in aqueous solutions. Within the limits of experimental errors both types of spectra are identical. The spectra of DNA-bound AMHA show a characteristic gap of ca. 3500 cm−1 between the 0-0-transitions of the long wave length 1La absorption and the fluorescence. AMHA molecules dissolved in the polar solvent water have a gap of even 4100 cm−1. This energy gap shows that the electron distribution of AMHA is strongly changed by light absorption and emission.Finally, using absorption spectroscopy, we investigated the binding of AMHA to DNA in aqueous solutions over a wide range of concentrations of the dye, of nuceleic acid (calf thymus), and of the competitor NaCl respectively. The Scatchard binding isotherms were determined. With the method of competitive salt effect three different bonds of AMHA to DNA can be distinguished even at low dye concentrations: The intercalation 1 of the fluorochrome F, binding constant KF1=1,1·105M−1, binding parameter n1=0,15; the pre-intercalative or external binding 2, KF2=6,9·105M−1, n2=0,21; the external binding 3, KF3=2,8·105M−1, n3=0,55. Externally bound dye molecules 2 and 3 occupy two phosphodiester residues of the DNA. A detailed discussion of the data and the competitive salt effect shows that in living cells only intercalated and small amounts of pre-intercalatively bound molecules 1 and 2 exist. The binding constant KF1=1,1·105M−1 of AMHA is unusual high in comparison with the constants of intercalation of other dyes, KF1=(1–4)·104M−1. Therefore, the amount of intercalated AMHA is also relatively high, and it is possible to visualize the DNA-bound fluorochrome in the nuclei and chromosomes of the living cells under the fluorescence microscope.The fluorochrome AMHA (3-amino-6-methoxy-9-(2-hydroxyethylamino)acridine) stains the nuclear chromatin and the chromosomes of living HeLa cells. At relatively low dye concentrations CF less than or equal to 10(-4) M and short incubation periods tI less than or equal to 2 h cell growth is not affected by the drug. But at higher CF and longer tI the population doubling time of the cell cultures rapidly increases, and finally the cells die. In vital staining experiments the dye AMHA preferentially binds to the DNA of the nuclei and to the chromosomes of the cells, respectively. The dye binding to DNA has been proved by the absorption and emission microspectra of the stained cells, and by the comparison with authentic spectra of AMHA bound to DNA in aqueous solutions. Within the limits of experimental errors both types of spectra are identical. The spectra of DNA-bound AMHA show a characteristic gap of ca. 3500 cm-1 between the 0-0-transitions of the long wave length 1La absorption and the fluorescence. AMHA molecules dissolved in the polar solvent water have a gap of even 4100 cm-1. This energy gap shows that the electron distribution of AMHA is strongly changed by light absorption and emission. Finally, using absorption spectroscopy, we investigated the binding of AMHA to DNA in aqueous solutions over a wide range of concentrations of the dye, of nucleic acid (calf thymus), and of the competitor NaCl respectively. The Scatchard binding isotherms were determined. With the method of competitive salt effect three different bonds of AMHA to DNA can be distinguished even at low dye concentrations: The intercalation 1 of the fluorochrome F, binding constant KF1 = 1.1.10(5) M-1, binding parameter n1 = 0.15; the pre-intercalative or external binding 2, KF2 = 6.9.10(5) M-1, n2 = 0.21; the external binding 3, KF3 = 2.8.10(5) M-1, n3 = 0.55. Externally bound dye molecules 2 and 3 occupy two phosphodiester residues of the DNA. A detailed discussion of the data and the competitive salt effect shows that in living cells only intercalated and small amounts of pre-intercalatively bound molecules 1 and 2 exist. The binding constant KF1 = 1.1.10(5) M-1 of AMHA is unusual high in comparison with the constants of intercalation of other dyes, KF1 = (1-4).10(4) M-1.(ABSTRACT TRUNCATED AT 400 WORDS)

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

Three new acridine dyes, 3-dimethylamino-6-methoxyacridine 1, 3-amino-6-methoxyacridine 2 and 3-amino-7-methoxyacridine 3, have been prepared and tested as fluorochromes of LM- and HeLa-cells. The dyes are basic compounds (pKA: 1 8,76; 2 8,01; 3 7,65) and form cations in neutral or acidic aqueous solutions by addition of a proton to the aza-nitrogen atom of the heterocycle. The fluorochromes stain fixed LM- and HeLa-cells at pH = 6. The fluorescence shows metachromasy similar to the staining with acridine orange AO according to the technique of Bertalanffy. But there is less fading of the fluorescence. The dye 1 is the most suitable fluorochrome of the series. It was studied in detail. Using optimized staining conditions the fluorescence of the nucleus is yellow-green that of the cytoplasm and the nucleoli orange or brownish-red. Enzymatic digestion experiments show that the dye cations are bound to DNA in the nucleus and to RNA in the cytoplasm or nucleoli. The absorption and emission spectra of the stained cells have been studied by means of microspectrophotometry. The absorption spectra of the nucleus and the cytoplasm are very similar. The maximum of the long wave length absorption of both occurs at 21400 cm-1 (467 nm) with a shoulder at ca 20100 cm-1 (498 nm). The fluorescence spectra of nucleus and cytoplasm of metachromatically stained cells are different. The emission maximum of the cytoplasm and nucleoli, 16200 cm-1 (617 nm), is red-shifted relative to the maximum of the nucleus, 18200 cm-1 (549 nm). This shift causes the metachromatic fluorescence effect. In addition we studied the concentration dependence of the absorption and fluorescence spectra of the cation 1 in aqueous solution, pH = 6, in the concentration range 6 X 10(-6)-6 X 10(-4) M. Shape and maximum of the long wave length absorption and emission depend only slightly on the concentration: Mean value of absorption maximum ca 21500 cm-1 (465 nm), shoulder at ca 20300 cm-1 (493 nm), fluorescence maximum ca 18300 cm-1 (547 nm). With growing concentration diminishes the molar absorptivity. This decrease in absorptivity and isosbestic points in the absorption spectra indicate the formation of dimers with growing dye concentration. The absorption spectra of the metachromatically stained cells and of the dye in aqueous solution are very similar.(ABSTRACT TRUNCATED AT 400 WORDS)SummaryThree new acridine dyes, 3-dimethylamino-6-methyoxyacridine 1, 3-amino-6-methoxyacridine 2 and 3-amino-7-methoxyacridine 3, have been prepared and tested as fluorochromes of LM- and HeLa-cells. The dyes are basic compounds (pKA: 1 8,76; 2 8,01; 3 7,65) and form cations in neutral or acidic aqueous solutions by addition of a proton to the aza-nitrogen atom of the heterocycle. The fluorochromes stain fixed LM- and HeLa-cells at pH=6. The fluorescence shows metachromasy similar to the staining with acridine orange AO according to the technique of Bertalanffy. But there is less fading of the fluorescence. The dye 1 is the most suitable fluorochrome of the series. It was studied in detail.Using optimized staining conditions the fluorescence of the nucleus is yellow-green that of the cytoplasm and the nucleoli orange or brownish-red. Enzymatic digestion experiments show that the dye cations are bound to DNA in the nucleus and to RNA in the cytoplasm or nucleoli.The absorption and emission spectra of the stained cells have been studied by means of microspectrophotometry. The absorption spectra of the nucleus and the cytoplasm are very similar. The maximum of the long wave length absorption of both occurs at 21400 cm−1 (467 nm) with a shoulder at ca 20100 cm−1 (498 nm).The fluorescence spectra of nucleus and cytoplasm of metachromatically stained cells are different. The emission maximum of the cytoplasm and nucleoli, 16200 cm−1 (617 nm), is red-shifted relative to the maximum of the nucleus, 18200 cm−1 (549 nm). This shift causes the metachromatic fluorescence effect.In addition we studied the concentration dependence of the absorption and fluorescence spectra of the cation 1 in aqueous solution, pH=6, in the concentration range 6×10−6–6×10−4 M. Shape and maximum of the long wave length absorption and emission depend only slightly on the concentration: Mean value of absorption maximum ca 21500 cm−1 (465 nm), shoulder at ca 20300 cm−1 (493 nm), fluorescence maximum ca 18300 cm−1 (547 nm). With growing concentration diminishes the molar absorptivity. This decrease in absorptivity and isosbestic points in the absorption spectra indicate the formation of dimers with growing dye concentration.The absorption spectra of the metachromatically stained cells and of the dye in aqueous solution are very similar. A careful comparison of the spectra make it probable that the dye cations bound to DNA in the nucleus are monomer and to RNA in the cytoplasm or nucleoli are dimer. The same has been observed with AO. But our spectral changes are smaller.The fluorescence of the dye cations bound to RNA in the cytoplasm is strongly red-shifted compared to the fluorescence of the nucleus. Absorption and emission spectra of metachromatically stained cytoplasm can be explained on the assumption that the RNA bound dye forms dimers D in the ground state and excimers E in the first excited state. Compared with D the excimers are stabilized and the bond distance R between the molecules is shortened. The potential energy curves V(R) of the ground state and the first excited state are discussed in detail. Accordingly D can only be observed in absorption and E in fluorescence. Our experimental results agree with the excimer hypothesis.Absorption, fluorescence and absorption polarisation spectra of 1 (cation and free base) have been measured in rigid ethanol at 77 K. The spectra are compared with quantum mechanical calculations in SCF-CI-PPP-approximation. According to that the two absorption bands of the free cation in aqueous solution at ca 20300 and 21500 cm−1 and of the bound cations at ca 20100 and 21400 cm−1 are classified as 0-0- and 0-1-transitions of the long wave length absorption of 1.ZusammenfassungDrei neue Acridinfarbstoffe, 3-Dimethylamino-6-methoxyacridin 1, 3-Amino-6-methoxyacridin 2 und 3-Amino-7-methoxyacridin 3 wurden synthetisiert und ihre Eigenschaften als Fluorochrome für LM- und HeLa-Zellen untersucht. Die Substanzen sind Basen (pKA: 1 8,76; 2 8,01; 3 7,65), die in neutralem und in saurem wäßrigem Medium Kationen bilden durch Protonierung des Aza-Stickstoffatoms vom Heterocyclus. Die Fluorochrome färben fixierte LM- und HeLa-Zellen bei pH=6. Die Fluoreszenz zeigt Metachromasie in Analogie zur Färbung nach Bertalanffy mit Acridinorange AO. Die Färbung hat höhere Photostabilität als bei AO. Die besten Eigenschaften als Fluorochrom hat 1, das im Detail untersucht wurde.Die Vorschrift der Färbung wurde optimiert. Kerne gefärbter Zellen fluoreszieren gelb-grün, Cytoplasma und Nucleoli orange bis braun-rot. Enzymatische Abbauversuche zeigen, daß der Farbstoff in den Kernen an DNA, im Cytoplasma bzw. den Nucleoli an RNA gebunden ist.Die Absorptions- und Emissionsspektren der fluorochromierten Zellen wurden mikrospektralphotometrisch untersucht. Die Absorptionsspektren von Kern und Cytoplasma sind sehr ähnlich. Das Maximum der längstwelligen Absorptionsbande beobachtet man bei beiden bei 21400 cm−1 (467 nm) mit einer Schulter bei ca. 20100 cm−1 (498 nm).Die Fluoreszenzspektren von Kern und Cytoplasma metachromatisch gefärbter Zellen sind verschieden. Das Maximum der Emission des Cytoplasmas bzw. der Nucleoli, 16200 cm−1 (617 nm), ist langwellig gegenüber dem Fluoreszenzmaximum des Kerns, 18200 cm−1 (549 nm), verschoben. Diese Verschiebung verursacht den metachromatischen Fluoreszenzeffekt.In Ergänzung wurde die Konzentrationsabhängigkeit der Absorptions- und Fluoreszenzspektren des Kations 1 in wäßriger Lösung, pH=6, im Konzentrationsintervall 6×10−6−6×10−4 M untersucht. Lage und Form der Banden hängen nur weinig von der Konzentration ab: Mittelwert des längstwelligen Absorptionsmaximums ca. 21500 cm−1 (465 nm), der langwelligen Schulter ca. 20300 cm−1 (493 nm) und des Fluoreszenzmaximums ca. 18300 cm−1 (547 nm). Mit steigender Konzentration nimmt der molare Extinktionskoeffizient ab. Diese Abnahme und isosbestische Punkte in den Absorptionsspektren weisen auf die Bildung von Dimeren mit steigender Konzentration hin.Die Absorptionsspektren der metachromatisch gefärbten Zellen und des Farbstoffs in Lösung sind sehr ähnlich. Ein sorgfältiger Vergleich der Spektren macht es wahrscheinlich, daß die DNA-gebundenen Farbstoffkationen im Kern monomer, die RNA-gebundenen im Cytoplasma bzw. den Nucleoli dimer sind. Ähnliches wurde bei AO gefunden. Die spektralen Effekte sind jedoch bei 1 viel kleiner.Die Fluoreszenz der RNA-gebundenen Farbstoffkationen des Cytoplasmas ist stark langwellig gegenüber der Kernfluoreszenz verschoben. Absorptions- und Emissionsspektren lassen sich unter der Annahme deuten, daß der RNA-gebundene Farbstoff des metachromatisch gefärbten Cytoplasmas bzw. der Nucleoli im Grundzustand Dimere D, im ersten angeregten Zustand Excimere E bildet. Die Excimeren sind gegenüber den Dimeren stabilisiert; der Bindungsabstand R zwischen den Molekülen ist verkürzt. Das Potentialschema V(R) für Grund- und ersten angeregten Zustand wird im Detail diskutiert. Danach kann D nur in Absorption, E nur in Emission beobachtet werden. Unsere experimentellen Befunde stehen mit einer Excimerenhypothese in Übereinstimmung.Absorptions-, Fluoreszenz- und Absorptionspolarisationsspektren von 1 (Kation und freie Base) wurden in glasartig erstarrtem Ethanol bei 77 K gemessen. Die Spektren wurden mit quantenmechanischen Modellrechnungen in SCF-CI-PPP-Approximation verglichen. Danach sind die beiden Absorptionsbanden des freien Kations in wäßriger Lösung bei ca. 20300 und 21500 cm−1 und des gebundenen Kations bei 20100 und 21400 cm−1 dem 0-0- und 0-1-Übergang der längstwelligen Elektronenbande von 1 zuzuordnen.

ber hydrophobe Acridinfarbstoffe zur Fluorochromierung von Mitochondrien in lebenden Zellen@@@Hydrophobic acridine dyes for fluorescence staining of mitochondria in living cells. 2. Comparison of staining of living and fixed HeLa-cells with NAO and DPPAO: 2. Mitteilung: Vergleich der Frbung lebender und fixierter HeLa-Zellen mit NAO und DPPAO

The hydrophobic fluorescence dyes NAO and DPPAO (see scheme of structural formulae) stain the mitochondria of living HeLa-cells. The trans-membrane potential favours the dye accumulation of the cation NAO and supports the hydrophobic interaction of the dye with the mitochondrial membrane lipids and proteins. The lecithin-like dye DPPAO is electrical neutral. Its binding to mitochondria of living cells is only caused by hydrophobic interaction. NAO and DPPAO stain also the mitochondria of glutaraldehyde fixed HeLa-cells in aqueous medium. Fluorescence staining occurs even after extraction of the lipids of the cell with acetone. We suppose that the dye accumulation in the mitochondria of the fixed cells is caused by the hydrophobic interaction between the dyes and the very hydrophobic mitochondrial lipids and proteins.SummaryThe hydrophobic fluorescence dyes NAO and DPPAO (see scheme of structural formulae) stain the mitochondria of living HeLa-cells. The trans-membrane potential favours the dye accumulation of the cation NAO and supports the hydrophobic interaction of the dye with the mitochondrial membrane lipids and proteins. The lecithinlike dye DPPAO is electrical neutral. Its binding to mitochondria of living cells is only caused by hydrophobic interaction. NAO and DPPAO stain also the mitochondria of glutaraldehyde fixed HeLa-cells in aqueous medium. Fluorescence staining occures even after extraction of the lipids of the cell with acetone. We suppose that the dye accumulation in the mitochondria of the fixed cells is caused by the hydrophobic interaction between the dyes and the very hydrophobic mitochondrial lipids and proteins.