Carbocyanine dyes stain the sarcoplasmic reticulum of beating heart cells

In search of a fluorescent dye suitable for monitoring membrane potentials of beating heart cells, we noticed that the carbocyanine dyes, CC₅ and CC₆, show a unique pattern of intracellular distribution in vital and glutaraldehyde-fixed cardiomyoblasts. This distribution is clearly different from that observed in fibroblasts. In heart cells, it parallels the localization of actin-myosin containing myofilaments as visualized by fluorescent antibody staining but it does not correspond to the localization of actin filaments or the microtubules. In fibroblasts these dyes stain only fine filaments and granules in the perinuclear space which correspond to the endoplasmic reticulum. This observation is evidence in support of the hypothesis that carbocyanine dyes accumulate selectively in the sarcoplasmic reticulum. It indicates that certain carbocyanine dyes may be useful tools to differentiate between muscle cells and connective tissue cells in cell cultures.     

Rational approach to select small peptide molecular probes labeled with fluorescent cyanine dyes for in vivo optical imaging

We demonstrate that the structure of carbocyanine dyes, which are commonly used to label small peptides for molecular imaging and not the bound peptide, controls the rate of extravasation from blood vessels to tissue. By examining several near-infrared (NIR) carbocyanine fluorophores, we demonstrate a quantitative correlation between the binding of a dye to albumin, a model plasma protein, and the rate of extravasation of the probe into tissue. Binding of the dyes was measured by fluorescence quenching of the tryptophans in albumin and was found to be inversely proportional to the rate of extravasation. The rate of extravasation, determined by kurtosis from longitudinal imaging studies using rodent ear models, provided a basis for quantitative measurements. Structure-activity studies aimed at evaluating a representative library of NIR fluorescent cyanine probes showed that hydrophilic dyes with binding constants several orders of magnitude lower than their hydrophobic counterparts have much faster extravasation rate, establishing a foundation for rational probe design. The correlation provides a guideline for dye selection in optical imaging and a method to verify if a certain dye is optimal for a specific molecular imaging application.     

Fluorescent carbocyanine dyes allow living neurons of identified origin to be studied in long-term cultures

A prerequisite for many studies of neurons in culture is a means of determining their original identity. We needed such a technique to study the interactions in vitro between a class of spinal cord neurons, sympathetic preganglionic neurons, and their normal target, neurons from the sympathetic chain. Here, we describe how we use two highly fluorescent carbocyanine dyes, which differ in color but are otherwise similar, to identify neurons in culture. The long carbon chain carbocyanine dyes we use are lipid-soluble and so become incorporated into the plasma membrane. Neurons can be labeled either retrogradely or during dissociation. Some of the labeled membrane gradually becomes internalized and retains its fluorescence, allowing identification of cells for several weeks in culture. These dyes do not affect the survival, development, or basic physiological properties of neurons and do not spread detectably from labeled to unlabeled neurons. It seems likely that cells become retrogradely labeled mainly by lateral diffusion of dye in the plane of the membrane. If so, carbocyanine dyes may be most useful for retrograde labeling over relatively short distances. An additional feature of carbocyanine labeling is that neuronal processes are brightly fluorescent for the first few days in culture, presumably because dye rapidly diffuses into newly inserted membrane. We have used carbocyanine dyes to identify sympathetic preganglionic neurons in culture. Our results indicate that preganglionic neurons can survive in the absence of their target cells and that several aspects of their differentiation in the absence of target appear normal.     

A quantitative resolution of the spectra of a membrane potential indicator,diS-C3-(5), bound to cell components and to red blood cells

Summary Cationic cyanine dyes have been widely used to measure electrical potentials of red blood cells and other membrane preparations. A quantitative analysis of the binding of the most extensively studied of these dyes, diS-C₃-(5), to red blood cells and their constituents is presented here. Absorption spectra were recorded for the dye in suspensions of isolated red cell membranes and in solutions of cell lysate. The dependence of the spectra on the concentrations of dye and cell constituents shows that the dye binds to these membranes as monomers with an absorbance maximum at 670 nm instead of 650 nm as for free aqueous dye and that the dye binds to oxyhaemoglobin partly as monomer but primarily as dimer, with absorbance maxima ca. 670 and 595 nm, respectively. Quantitative estimates are derived for all binding constants and extinction coefficients. These estimates are applied to suspensions of whole cells to predict the dye binding, absorbance spectra, and calibration curves of binding and fluorescencevs. membrane voltage. Satisfactory agreement is found with binding and absorbance data for whole cells at zero membrane potential and with the binding and fluorescence data reported by Hladky and Rink (J. Physiol. (London) 263:287, 1976) for cells driven to positive and negative potentials using valinomycin. The marked tendency of oxyhaemoglobin to bind dye as dimer is not shared by some other proteins tested, including deocyhaemoglobin and oxymyoglobin.     

Flow cytometric detection of membrane potential changes in murine lymphocytes induced by concanavalin A.

The effect of the mitogenic lectin concanavalin A on the membrane potential of murine lymphocytes was investigated by observing the fluorescence of cells stained with carbocyanine and oxonol dyes. We describe a rapid and reliable method for detecting lectin-induced membrane potential changes in individual cells by flow cytometric analysis of oxonol fluorescence. By 10 min after addition of lectin to suspensions of isolated cells from lymph node, 7-15% of the cells have responded by releasing oxonol dye, indicating a membrane hyperpolarization. The dose onset of this response is similar to that for mitogenesis, which was assessed by measuring [3H]thymidine incorporation. The effect is abolished by alpha-methyl mannoside (100mM), which prevents concanavalin A from binding to the cells, but not by fucose (100mM). When cells are treated with lectin in medium from which Ca2+ has been omitted or to which quinine (0.5mM) has been added, a membrane depolarization is observed. Since these are conditions under which activation of plasma membrane Ca2+-dependent K+ channels is prevented, these findings support the view that the early hyperpolarization of these cells is brought about by an increase in intracellular free [Ca2+].     

DFT and TDDFT study on the electronic structure and photoelectrochemical properties of dyes derived from cochineal and lac insects as photosensitizer for dye-sensitized solar cells

Essential parameters related to the photoelectrochemical properties, such as ground state geometries, electronic structures, oxidation potential and electron driving force, of cochineal insect dyes were investigated by DFT and TDDFT at the B3LYP/6-31+G(d,p) level of the theory. The results show that the major charge flow dynamic for all dyes is the HOMO→LUMO transition. The bi-coordinated binding mode, in which the dye uses one carboxyl- and hydroxyl oxygen bound to Ti(IV), is found for all dye-TiO₂ systems. Additionally, the doubly bi-coordinated binding mode in which the dye used both carboxyl groups bound to two Ti(IV) is also possible due to high energy distribution occupied at anchoring groups. This study highlights that most of these insect dyes can be good photosensitizers in dye-sensitized solar cells based on their strong binding to the TiO₂ surface, good computed excited state oxidation potential and thermodynamically favored electron driving force.     

Monitoring Changes in Membrane Polarity,Membrane Integrity,and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC₄(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca²⁺, K⁺, and H⁺, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.     

The cationic carbocyanine dyes Stains-all DBTC, and Ethyl-Stains-all, DBTC-3,3',9 triethyl.

A simple method is presented for distinguishing two closely related metachromatic carbocyanine dyes: Ethyl-Stains-all, a triethyl dye, and Stains-all, a diethyl methyl dye. This has become important since one lot of the triethyl dye was distributed erroneously under the diethyl methyl label. The dyes differ in solubility and in differential staining of macromolecules. Studies performed with both dyes are summarized.     

Fluorescence resonance energy transfer (FRET) and competing processes in donor-acceptor substituted DNA strands: a comparative study of ensemble and single-molecule data

We studied the fluorescence resonance energy transfer (FRET) efficiency of different donor-acceptor labeled model DNA systems in aqueous solution from ensemble measurements and at the single molecule level. The donor dyes: tetramethylrhodamine (TMR); rhodamine 6G (R6G); and a carbocyanine dye (Cy3) were covalently attached to the 5'-end of a 40-mer model oligonucleotide. The acceptor dyes, a carbocyanine dye (Cy5), and a rhodamine derivative (JA133) were attached at modified thymidine bases in the complementary DNA strand with donor-acceptor distances of 5, 15, 25 and 35 DNA-bases, respectively. Anisotropy measurements demonstrate that none of the dyes can be observed as a free rotor; especially in the 5-bp constructs the dyes exhibit relatively high anisotropy values. Nevertheless, the dyes change their conformation with respect to the oligonucleotide on a slower time scale in the millisecond range. This results in a dynamic inhomogeneous distribution of donor/acceptor (D/A) distances and orientations. FRET efficiencies have been calculated from donor and acceptor fluorescence intensity as well as from time-resolved fluorescence measurements of the donor fluorescence decay. Dependent on the D/A pair and distance, additional strong fluorescence quenching of the donor is observed, which simulates lower FRET efficiencies at short distances and higher efficiencies at longer distances. On the other hand, spFRET measurements revealed subpopulations that exhibit the expected FRET efficiency, even at short D/A distances. In addition, the measured acceptor fluorescence intensities and lifetimes also partly show fluorescence quenching effects independent of the excitation wavelength, i.e. either directly excited or via FRET. These effects strongly depend on the D/A distance and the dyes used, respectively. The obtained data demonstrate that besides dimerization at short D/A distances, an electron transfer process between the acceptor Cy5 and rhodamine donors has to be taken into account. To explain deviations from FRET theory even at larger D/A distances, we suggest that the pi-stack of the DNA double helix mediates electron transfer from the donor to the acceptor, even over distances as long as 35 base pairs. Our data show that FRET experiments at the single molecule level are rather suited to resolve fluorescent subpopulations in heterogeneous mixture, information about strongly quenched subpopulations gets lost.     

The interaction of methylene blue,azure B,and thionine with DNA: Formation of complexes with polynucleotides and mononucleotides as model systems

The interactions of methylene blue, azure B, and thionine with calf thymus DNA, [poly (dG-dC)]₂, [poly(dA-dT)]₂, and the constituent mononucleotides 2′-deoxyguanosine-5′-monophosphate(dGMP), 2′-deoxyadenosine-5′-monophosphate(dAMP), 2′-deoxycytidine-5′-monophosphate(dCMP), and thymidine-5′-monophosphate(dTMP) have been studied by steady-state absorption spectroscopy and with equilibrium dialysis. Scatchard plots for binding of the dyes to the nucleic acid polymers were convex downward at low binding ratios, characteristic of intercalation, and binding constants for this mode were calculated under conditions of varying ionic strength. For each of the dyes, binding constants with [poly(dG-dC)]₂ and [poly(dA-dT)]₂ were of the same order of magnitude, so that previously reported (G-C) preferentially is not very marked. At high binding ratios, the Scatchard plots did not return to the abscissa but curved upward, indicative of a weaker cooperative binding mode, occurring under conditions where the dye is in excess, which is suggested to be external stacking of the dye molecules promoted by the polyanion. The dependence of the absorption spectra on added salt demonstrated a shift in the strong binding mode for the three dyes with [poly(dA-dT)]₂ with increasing ionic strength, while with [poly(dG-dC)]₂ this does not occur. The dyes were found to bind to purine but not pyrimidine mononucleotides with dGMP and dAMP, 1:1 complexes were formed initially and also 1:2 dye/nucleotide complexes with increasing nucleotide concentrations. Under low salt conditions, binding to dAMP was slightly stronger than to dGMP for the three dyes studied, while at high ionic strength, when the binding constants are significantly lower, all binding constants become very similar. Binding to mononucleotides is suggested to be primarily stabilised by π-π stacking interactions between the planar dyes and the nucleobases: for thionine and azure B there also appears to be H-bonds between the exocyclic amines and the sugar–phosphates conferring extra stability. Neither increasing the number of phosphate groups on the nucleotides nor changing from deoxyribose to ribose sugars had any significant effect on the binding constants. © 1995 John Wiley & Sons, Inc.     

Comparison of spectrum-shifting intracellular pH probes 5'(and 6')-carboxy-10-dimethylamino-3-hydroxyspiro[7H-benzo[c]xanthene-7, 1'(3'H)-isobenzofuran]-3'-one and 2',7'-biscarboxyethyl-5(and 6)-carboxyfluorescein.

The dyes carboxy-SNARF-1 and BCECF are fluorescent probes of intracellular pH that exhibit changes in spectral shape upon proton binding which allow one to use measurements of fluorescence at two or more wavelengths in order to measure pH without artifacts associated with variability in dye loading, etc. In evaluating these dyes for this study, whole spectra, rather than measurements at two wavelengths, were analyzed. For BCECF, the effects of the intracellular milieu were minimal: both the pH-sensitive excitation spectrum and the pKa agreed closely with values found in extracellular solution. In contrast, both the spectra and the pKa for the emission spectrum-shifting carboxy-SNARF-1 showed significant differences between intracellular and extracellular dye. As a result, extremely misleading values for intracellular pH will be obtained if one attempts to use extracellular dye to calibrate intracellular carboxy-SNARF-1 measurements. Multiple origins were found for the discrepancy: (i) the intracellular dye was found to be significantly quenched, with the deprotonated form being more strongly quenched than the protonated form; and (ii) the pKa for the equilibrium with intracellular hydrogen ions was shifted by +0.2 pH units. These effects were readily reversed by disruption of the cell, but were not due to sequestering of dye in an acidic cell compartment.     

Calculation of binding length of base-specific DNA dyes by comparison of sequence and flow cytometric data. Application to Oryza sativa and Arabidopsis thaliana

From biochemical experiments it has been found that AT- and GC-specific dyes need a certain number of consecutive bases of the same type for binding one dye molecule. From known base sequences the amount of bases included in dye binding can be calculated and compared with experimental data from flow cytometry. Oryza sativa and Arabidopsis thaliana are the first higher plants which are nearly completely (>90%) sequenced. From the published sequences the theoretical fluorescence intensity of base-specific dyes in relation to a base-unspecific dye is calculated for different binding lengths. These values are compared with the actual fluorescence intensities of nuclei analyzed by flow cytometry. For all investigated dyes (DAPI, Hoechst 33258, Hoechst 33342 (all AT specific) and Mithramycin A (GC specific)) a binding length of 1 bp results from the comparison of theoretical and experimental data. This is, however, in disagreement with former results on dye binding. The main reason for the discrepancy seems to be the remaining gap in the sequencing of the Arabidopsis genome.     

Membrane potential can be determined in individual cells from the nernstian distribution of cationic dyes.

The distribution of a selection of cationic fluorescent dyes can be used to measure the membrane potential of individual cells with a microfluorometer. The essential attributes of these dyes include membrane permeability, low membrane binding, spectral properties which are insensitive to environment, and, of course, strong fluorescence. A series of dyes were screened on HeLa cells for their ability to meet these criteria and several commercially available dyes were found to be satisfactory. In addition, two new dyes were synthesized for this work by esterification of tetramethyl rhodamine. The analysis of the measured fluorescent intensities requires correction for fluorescence collected from outside the plane of focus of the cell and for nonpotentiometric binding of the dye. The measurements and analysis were performed on three different cell types for which there exists a body of literature on membrane potential; the potentials determined in this work were always within the range of literature values. The rhodamine esters are nontoxic, highly fluorescent dyes which do not form aggregates or display binding-dependent changes in fluorescence efficiency. Thus, their reversible accumulation is quantitatively related to the contrast between intracellular and extracellular fluorescence and allows membrane potentials in individual cells to be continuously monitored.     

G-quadruplex DNA binding by a series of carbocyanine dyes.

We have examined a number of carbocyanine dyes for their ability to bind intramolecular G-quadruplex DNA structures (G4'-DNA) using a Taq polymerase stop assay. Of the five dyes examined, only one, N,N'-diethylthiacarbocyanine iodide (DTC), was found to bind to G4'-DNA. DTC was also the only dye found to inhibit human telomerase at 50 microM concentration.     

Biophysical Characterization of Styryl Dye-Membrane Interactions

Styryl dyes (also referred to as FM dyes) become highly fluorescent upon binding to membranes and are often used to study synaptic vesicle recycling in neurons. To date, however, no direct comparisons of the fluorescent properties, or time-resolved (millisecond) measurements of dye-membrane binding and unbinding reactions, for all members of this family of probes have been reported. Here, we compare the fluorescence intensities of each member of the FM dye family when bound to membranes. This analysis included SGC5, a new lipophilic fluorescent dye with a unique structure. Fluorescence intensities depended on the length of the lipophilic tail of each dye, with a rank order as follows: SGC5 > FM1-84 > FM1-43 > SynaptoGreen C3 > FM2-10/FM4-64/FM5-95. Stopped-flow measurements revealed that dye hydrophobicity determined the affinity and departitioning rates for dye-membrane interactions. All of the dyes dissociated from membranes on the millisecond timescale, which is orders of magnitude faster than the overall destaining rate (timescale of seconds) of these dyes from presynaptic boutons. Departitioning kinetics were faster at higher temperatures, but were unaffected by pH or cholesterol. The data reported here aid interpretation of dye-release kinetics from single synaptic vesicles, and indicate that these probes dissociate from membranes on more rapid timescales than previously appreciated.     

Effect of chemical structures of acridine and triphenylmethane dyes on the induced optical activity of DNA-dye complexes

Fifteen symmetrically substituted acridine dyes, all of which are interrelated by their chemical structures, each belonging to a C_2v symmetry, and three triphenylmethane dyes with amino or dimethylamino substituents are utilized to study necessary conditions for the appearance of extrinsic Cotton effects upon their binding to native and heat-denatured deoxyribonucleic acid (DNA). Three different kinds of the DNA–dye complexes, i.e., (1) dye added to native DNA, (2) heat-denatured DNA–dye complex, and (3) dye added to preheated DNA, were examined for each dye at a fixed P/D value of about 4. Optical activity was always observed for the compelexes of type (1) in each absorption band of the dyes in the visible and near-ultraviolet region. Two exceptions are 9-acetamido- and 9-hydroxyacridine, both being nonionic in aqueous solution at a pH range of 6. Acridinium chloride was unable to exhibit any definite extrinsic Cotton effect for complexes (2) and (3). Thus, the monocationic form of a dye due to the protonation or quaternization of the ring nitrogen in acridines or exonuclear amino nitrogen in triphenylmethane dyes is concluded to be an essential factor for extrinsic Cotton effect to appear. Changes in the absorption spectra upon complex formation are also related to the structure of dyes. Hypochromism and bathochromism are associated with the induced optical activity in all cases in the presence of native and denatured DNA.     

Inter- and intramolecular fluorescence quenching of organic dyes by tryptophan

Steady-state and time-resolved fluorescence measurements were performed to elucidate the fluorescence quenching of oxazine, rhodamine, carbocyanine, and bora-diaza-indacene dyes by amino acids. Among the natural amino acids, tryptophan exhibits the most pronounced quenching efficiency. Especially, the red-absorbing dyes ATTO 655, ATTO 680, and the oxazine derivative MR 121 are strongly quenched almost exclusively by tryptophan due to the formation of weak or nonfluorescent ground-state complexes with association constants, K(ass.), ranging from 96 to 206 M(-1). Rhodamine, fluorescein, and bora-diaza-indacene derivatives that absorb at shorter wavelengths are also quenched substantially by tyrosine residues. The quenching of carbocyanine dyes, such as Cy5, and Alexa 647 by amino acids can be almost neglected. While quenching of ATTO 655, ATTO 680, and the oxazine derivative MR121 by tryptophan is dominated by static quenching, dynamic quenching is more efficient for the two bora-diaza-indacene dyes Bodipy-FL and Bodipy630/650. Labeling of the dyes to tryptophan, tryptophan-containing peptides, and proteins (streptavidin) demonstrates that knowledge of these fluorescence quenching processes is crucial for the development of fluorescence-based diagnostic assays. Changes in the fluorescence quantum yield of dye-labeled peptides and proteins might be used advantageously for the quantification of proteases and specific binding partners.     

Microbial decolorization and degradation of synthetic dyes: a review

The synthesis of dyes and pigments used in textiles and other industries generate the hazardous wastes. A dye is used to impart color to materials of which it becomes an integral part. The waste generated during the process and operation of the dyes commonly found to contain the inorganic and organic contaminant leading to the hazard to ecosystem and biodiversity causing impact on the environment. The amount of azo dyes concentration present in wastewater varied from lower to higher concentration that lead to color dye effluent causing toxicity to biological ecosystem. The physico-chemical treatment does not remove the color and dye compound concentration. The decolorization of the dye takes place either by adsorption on the microbial biomass or biodegradation by the cells. Bioremediation takes place by anaerobic and/or aerobic process. The anaerobic process converts dye in toxic amino compounds which on further treatment with aerobic reaction convert the intermediate into CO₂ biomass and inorganics. In the present review the decolorization and degradation of azo dyes by fungi, algae, yeast and bacteria have been cited along with the anaerobic to aerobic treatment processes. The factors affecting decolorization and biodegradation of azo dye compounds such as pH, temperature, dye concentration, effects of CO₂ and Nitrogen, agitation, effect of dye structure, electron donor and enzymes involved in microbial decolorization of azo dyes have been discussed. This paper will have the application for the decolorization and degradation of azo dye compound into environmental friendly compounds.     

Characterization of a carbocyanine derivative as a fluorescent penetration probe

The fluorescent carbocyanine dye 3,3-diheptyloxycarbocyanine [DiOC7(3)], originally described as a membrane potential probe, penetrates poorly into multicell spheroids. Since the dye is retained in the cells following spheroid disaggregation, cells can be selected from different depths within the spheroid using fluorescence-activated cell sorting. Characterization of the binding kinetics, stability, and toxicity of this probe were undertaken, and intercompared with Hoechst 33342. The optimum drug dose for achieving good separation of internal and external cells of spheroids is about tenfold lower than for Hoechst 33342, and like Hoechst, DiOC7(3) is toxic at concentrations at least tenfold higher than those required to produce a good gradient for cell separation. When cells are removed from the stain, cellular fluorescence decreases to half the initial intensity within 2 hours; however, unlike Hoechst, the carbocyanine dye does not transfer between cells.     

On the Origin of the Stronger Binding of PIB over Thioflavin T to Protofibrils of the Alzheimer Amyloid-β Peptide: A Molecular Dynamics Study

Pittsburgh compound B (PIB) is a neutral derivative of the fluorescent dye Thioflavin T (ThT), which displays enhanced hydrophobicity and binding affinity to amyloid fibrils. We present molecular dynamics simulations of binding of PIB and ThT to a common cross-β-subunit of the Alzheimer Amyloid-β peptide (Aβ). Our simulations of binding to Aβ_9-40 protofibrils show that PIB, like ThT, selectively binds to the hydrophobic or aromatic surface grooves on the β-sheet surface along the fibril axis. The lack of two methyl groups and charge in PIB not only improves its hydrophobicity but also leads to a deeper insertion of PIB compared to ThT into the surface grooves. This significantly increases the steric, aromatic, and hydrophobic interactions, and hence leads to stronger binding. Simulations on protofibrils consisting of the more-toxic Aβ_17-42 revealed an additional binding mode in which PIB and ThT insert into the channel that forms in the loop region of the protofibril, sandwiched between two sheet layers. Our simulations indicate that the rotation between the two ring parts of the dyes is significantly more restricted when the dyes are bound to the surface of the cross-β-subunits or to the channel inside the Aβ_17-42 cross-β-subunit, compared with free solution. The specific conformations of the dyes are influenced by small chemical modifications (ThT versus PIB) and by the environment in which the dye is placed.