Cultivation of marine shrimp in biofloc technology (BFT) system under different water alkalinities.

The aim of this study was to evaluate the influence of different levels of alkalinity for the superintensive cultivation of marine shrimp Litopenaeus vannamei in biofloc system. A total of 12 experimental circular units of 1000L were used supplied with 850L water from a nursery, populated at a density of 165 shrimps.m-3 and average weight of 5.6 g. The treatments, in triplicate, consisted in four levels of alkalinity in the water: 40, 80, 120 and 160 mg.L-1 of calcium carbonate. To correct the alkalinity was used calcium hydroxide (CaOH). It was observed a decrease in pH of the water in the treatments with lower alkalinity (p<0.05). The total suspended settleable solids were also lower in the treatment of low alkalinity. No significant difference was observed in other physico-chemical and biological parameters in the water quality assessed, as well as the zootechnical parameters of cultivation between treatments (p≥0.05). The results of survival and growth rate of shrimps were considered suitable for the cultivation system used in the different treatments. The cultivation of marine shrimp Litopenaeus vannamei in biofloc at density of 165 shrimps.m-3 can be performed in waters with alkalinity between 40 and 160 mg.L-1 of CaCO3, without compromising the zootechnical indexes of cultivation.

Cultivation of marine shrimp in biofloc technology (BFT) system under different water alkalinities / Cultivo de camarões marinhos em sistema de bioflocos (BFT) sob diferentes alcalinidades da água

AbstractThe aim of this study was to evaluate the influence of different levels of alkalinity for the superintensive cultivation of marine shrimp Litopenaeus vannamei in biofloc system. A total of 12 experimental circular units of 1000L were used supplied with 850L water from a nursery, populated at a density of 165 shrimps.m–3 and average weight of 5.6 g. The treatments, in triplicate, consisted in four levels of alkalinity in the water: 40, 80, 120 and 160 mg.L–1 of calcium carbonate. To correct the alkalinity was used calcium hydroxide (CaOH). It was observed a decrease in pH of the water in the treatments with lower alkalinity (p<0.05). The total suspended settleable solids were also lower in the treatment of low alkalinity. No significant difference was observed in other physico-chemical and biological parameters in the water quality assessed, as well as the zootechnical parameters of cultivation between treatments (p≥0.05). The results of survival and growth rate of shrimps were considered suitable for the cultivation system used in the different treatments. The cultivation of marine shrimp Litopenaeus vannamei in biofloc at density of 165 shrimps.m–3 can be performed in waters with alkalinity between 40 and 160 mg.L–1 of CaCO3, without compromising the zootechnical indexes of cultivation.

ResumoO objetivo deste trabalho foi avaliar a influência de diferentes níveis de alcalinidade para o cultivo do camarão marinho Litopenaeus vannamei em sistema superintensivo em bioflocos. Foram utilizadas 12 unidades experimentais circulares de 1000L abastecido com 850L de água provenientes de um berçário intensivo, povoadas a uma densidade de 165 camarões.m-3 e peso médio 5,6g. Os tratamentos em triplicata consistiram de quatro níveis de alcalinidade na água: 40, 80, 120 e 160 mg.L–1 de carbonato de cálcio. Para correção da alcalinidade, foi utilizado cal hidratada (CaOH). Foi observado um decréscimo no pH da água nos tratamentos de menor alcalinidade (p<0,05). Os sólidos suspensos sedimentáveis totais também foram menores nos tratamentos de menor alcalinidade. Não foi observada diferença significativa nos demais parâmetros físico-químicos e biológicos de qualidade de água avaliados, assim como nos parâmetros zootécnicos do cultivo entre os tratamentos (p≥0,05). Os resultados de sobrevivência e taxa de crescimento dos camarões foram considerados adequados para o sistema de cultivo utilizado nos distintos tratamentos. O cultivo do camarão marinho Litopenaeus vannamei em bioflocos na densidade de 165 camarões.m–3 pode ser realizado em águas com alcalinidade entre 40 a 160 mg.L–1 de CaCO3, sem comprometer os índices zootécnicos do cultivo.

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.

Romanowsky dyes and Romanowsky-Giemsa effect. 5. Structural investigations of the purple DNA-AB-EY dye complexes of Romanowsky-Giemsa staining.

A 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 approximately 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 oriented 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.(ABSTRACT TRUNCATED AT 400 WORDS)

[Model investigations on the structure of the purple dye complex of Giemsa staining]. / Modelluntersuchungen zur Struktur des purpurnen Farbstoffkomplexes der Giemsa-Färbung.

Nuclei of Giemsa stained cells show a purple coloration, which is generated by a complex of DNA, azure B (AB) and eosin Y (EY). The structure of this complex is unknown. Its absorption spectrum shows a sharp and strong band at 18,100 cm-1 (552 nm), the so called Romanowsky band (RB). It is possible to produce the complex outside of the cell, but it is cubersome to handle. Easier to handle is a purple complex composed of chondroitin sulfate (CHS), AB and EY, which also shows a sharp and strong RB at 18,100 cm-1 in the absorption spectrum. This CHS-AB-EY complex is a model for the DNA-AB-EY complex of Giemsa stained cell nuclei. We tried to investigate its structure. In the first step of the staining procedure CHS binds AB cations forming a stable CHS-AB complex. In the case of saturation each anionic SO4- and COO- -binding site of CHS is occupied by one dye cation and the complex has 1:1 composition. It has a strong and broad absorption band with its maximum at ca. 18,000 cm-1 (556 nm). In the second step the CHS-AB complex additionally binds EY dianions forming the purple CHS-AB-EY complex with its RB at 18,100 cm-1. This band can be clearly distinguished from the broad absorption of the bound AB cations. RB is generated by the EY chromophore, whose absorption is shifted to longer wavelength by the interaction with the CHS-AB framework.

[Spectroscopic and thermodynamic investigations on the binding of azure B to chondroitin sulfate and the structure of the metachromatic dye complex]. / 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)

[Romanowsky dyes and the Romanowsky-Giemsa effect. 3. Microspectrophotometric studies of Romanowsky-Giemsa staining. Spectroscopic evidence of a DNA-azure B-eosin Y complex producing the Romanowsky-Giemsa effect]. / Uber Romanowsky-Farbstoffe und den Romanowsky-Giemsa-Effekt. 3. Mitteilung: Mikrospektralphotometrische Untersuchung der Romanowsky-Giemsa-Färbung. Spektroskopischer Nachweis eines DNA-Azur B-Eosin Y-Komplexes, der den Romanowsky-Giemsa-Effekt verursacht.

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)

[Bertalanffy-like fluorescence staining with 3-dimethylamino-6-methoxyacridine]. / Uber 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)

[Hydrophobic acridine dyes for fluorescence staining of mitochondria in living cells. 1. Thermodynamic and spectroscopic properties of 10-n-alkylacridine orange chlorides]. / Uber hydrophobe Acridinfarbstoffe zur Fluorochromierung von Mitochondrien in lebenden Zellen. 1. Mitteilung: Thermodynamische und spektroskopische Eigenschaften von 10-n-Alkyl-acridiniumorange-chloriden.

10-n-Alkyl-acridine-orange-chlorides (alkyl-AOs) are excellent dyes for fluorescence staining of mitochondria in living cells. The thermodynamic and spectroscopic properties of the series alkyl = methyl to nonyl have been investigated. The dyes form dimers in aqueous solution. The dimerisation is mainly a consequence of the hydrophobic interaction. The dissociation constant K respectively association constant K-1 of the dimers describes the hydrophobic interaction and therefore the hydrophobic properties of the dye cations. The dissociation constant K = K0 at the standard temperature T = 298 K has been determined spectroscopically in aqueous solution. It depends on the length of the alkyl residue n-CmH2m + 1 (m = 1 - 9) (Table 2). In addition the standard dissociation enthalpies (energies) delta H0 and dissociation entropies delta S0 have been determined from the temperature dependence of K (Table 2). With increasing chain length m the thermodynamic parameters K0, delta H0, delta S0 decrease. Therefore with growing m the dimers are stabilized. This stabilization is an entropic effect which is diminished by the energetic effect. The change of the thermodynamic parameters with m is in agreement with the concept of hydrophobic interaction and the stabilization of water structure in the surroundings of hydrophobic residues. As one would expect nonyl-AO is the most hydrophobic dye of the series. As an example the spectroscopic properties of nonyl-AO have been determined. We measured the absorption, luminescence and polarization spectra in rigid ethanol at 77 K. Under these conditions alkyl-AOs associate like dyes in Water at room temperature. The spectra depend on the concentration of the solution. In very dilute solution we observe mainly the spectra of the monomers M, in concentrated solution the spectra of the dimers D. The spectra of M and D are characteristically different. The monomers have one long wave length absorption M1 = 20.000 cm-1 with resonance fluorescence. In addition there is a long living phosphorescence at 16.600 cm-1. Its polarization is nearly perpendicular to the plane of the AO residue. The dimers have two long wave length absorption bands D1 = 18.700 and D2 = 21.200 cm-1 with very different intensities. D1 has very low intensity and is forbitten, D2 is allowed. D1 shows fluorescence. Phosphorescence has not been observed. D1, D2 and also M1 are polarized in the plane of the AO residue. At short wave length absorption and polarization spectra are very similar. From the spectra we constructed the energy level diagram of M and D (Fig. 9). The first excited state of M splits in D in two levels. The level splitting and the transition i

The new and reproducible Papanicolaou stain. Morphologic and spectrophotometric observations on the influence of stain composition on staining results.

The morphologic and spectroscopic characteristics of a new and reproducible modification of the Papanicolaou stain are briefly described. The main features of this modification are (1) replacement of the natural dye hematoxylin by the synthetic and chemically well defined dye thionin, (2) introduction of an alcoholic counterstain consisting of eosin gamma and fast green FCF only and (3) employment of alcoholic solutions only. The absorption characteristics of hematoxylin and thionin bound to chromatin are influenced by the cytoplasmic counterstaining, especially by the two green dyes, the absorption peaks of which are close to those of the nuclear stains. The implications of these results for visual and automated cytologic diagnosis are briefly discussed.

[Romanowsky dyes and Romanowsky-Giemsa effect. 2. Eosin Y, erythrosin B, tetrachlorofluorescein, spectroscopic characterization of pure dyes, association of eosin Y]. / Uber Romanowsky-Farbstoffe und den Romanowsky-Giemsa-Effekt. 2. Mitteilung: Eosin Y, Erythrosin B, Tetrachlorfluoreszein. Specktroskopische Charakterisierung der reinen Farbstoffe, Assoziation von Eosin Y.

Analytically pure samples of the Romanowsky dyes eosin y, erythrosin b and tetrachlorofluorescein are prepared. DC of the dye samples shows no contaminations. We measured the absorption spectra of the dye dianions in alkaline aqueous solution and of the dye acids in 95% ethanol at very low dye concentrations. The molar extinction coefficients of the long wavelength absorption of the monomeric dye species are determined (Table 1). The extinction coefficients may be used for standardisation of dye samples. The absorption spectra of eosin y in aqueous solution are dependent on concentration. Using a new very sensitive method it was possible to identify two association equilibria from the concentration dependency of the spectra. Dimers are formed even in very dilute solutions, at higher concentrations tetramers. The dissociation constant of the dimers D in monomers M at 293 K, pH = 12, is K21 = 2,9 X 10(-5) M; of the tetramers Q in dimers D K42 = 2,4 X 10(-3) M. From the experimental spectra of eosin solutions at various concentrations, pH = 12, and the equilibrium constants K21, K42 the absorption spectra of the pure monomers, dimers and tetramers are calculated. M has one long wavelength absorption band, VM = 19300 cm-1, epsilon M = 1,03 X 10(5) M-1 cm-1; D also one absorption band, VD = 19300 cm-1, epsilon D = 1,74 X 10(5) M-1 cm-1; Q two absorption bands, VQ1 = 19100, VQ2 = 20200 cm-1, epsilon Q1 = 1,65 X 10(5), epsilon Q2 = 1,96 X 10(5) M-1 cm-1. The absorption spectrum of the dimers is discussed by quantum mechanics.

[Romanowsky dyes and romanowsky-Giemsa effect. 1. Azure B, purity and content of dye samples, association (author's transl)]. / Uber Romanowsky-Farbstoffe und den Romanowsky-Giemsa-Effekt. 1. Mitteilung: Azur B, Reinheit und Gehalt von Farbstoffen, Assoziation.

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.