Fluorescence labeling of mitochondria in living cells by the cationic photosensitizer ZnTM2,3PyPz, and the possible roles of redox processes and pseudobase formation in facilitating dye uptake.

The study of labeling selectivity and mechanisms of fluorescent organelle probes in living cells is of continuing interest in biomedical sciences. The tetracationic phthalocyanine-like ZnTM2,3PyPz photosensitizing dye induces a selective violet fluorescence in mitochondria of living HeLa cells under UV excitation that is due to co-localization of the red signal of the dye with NAD(P)H blue autofluorescence. Both red and blue signals co-localize with the green emission of the mitochondria probe, rhodamine 123. Microscopic observation of mitochondria was improved using image processing and analysis methods. High dye concentration and prolonged incubation time were required to achieve optimal mitochondrial labeling. ZnTM2,3PyPz is a highly cationic, hydrophilic dye, which makes ready entry into living cells unlikely. Redox color changes in solutions of the dye indicate that colorless products are formed by reduction. Spectroscopic studies of dye solutions showed that cycles of alkaline titration from pH 7 to 8.5 followed by acidification to pH 7 first lower, then restore the 640 nm absorption peak by approximately 90%, which can be explained by formation of pseudobases. Both reduction and pseudobase formation result in formation of less highly charged and more lipophilic (cell permeant) derivatives in equilibrium with the parent dye. Some of these are predicted to be lipophilic and therefore membrane-permeant; consequently, low concentrations of such species could be responsible for slow uptake and accumulation in mitochondria of living cells. We discuss the wider implications of such phenomena for uptake of hydrophilic fluorescent probes into living cells.

At least four distinct blue cationic phthalocyanine dyes sold as "alcian blue" raises the question: what is alcian blue?

We investigated physicochemical characteristics of dye lots sold as "alcian blue" using the Biological Stain Commission (BSC) precipitation test, differential scanning calorimetry, high performance liquid chromatography, thin layer chromatography and UV/visible spectroscopy. Four blue phthalocyanine dyes were detected in 11 commercial dye lots. These four included the original ingrain blue 1 CI 74000 dye and the dye sold with the name "alcian blue pyridine variant"; we discuss also the possible identity of the additional two dyes. A proposed extension to the BSC analytic scheme is presented that could distinguish three categories of commercial alcian blue dyes from each other and from the original alcian blue 8G.

Revised tests and standards for Biological Stain Commission certification of alcian blue dyes.

Alcian blue dyes are copper phthalocyanines with a variety of cationic side chains; they are useful for staining carbohydrate polyanions while avoiding staining of nucleic acids. The properties of the original alcian blue and of similar dyes with published chemical structures are reviewed here. Variation among samples submitted to the Biological Stain Commission (BSC) for certification has led to the recognition of two types of commercially available alcian blue at this time. The designation "alcian blue 8G or equivalent" is reserved for dyes that resemble alcian blue 8GX manufactured in the 1960s (CI 74240; ingrain blue 1). These dyes react with alkali to form an insoluble pigment that cannot be re-dissolved in acid. The name "alcian blue variant" is for similar dyes that do not form insoluble pigments; an alkali-induced precipitate, if formed, re-dissolves with acidification. For certification by the BSC, both types of alcian blue must dissolve in 3% acetic acid to make a 1% solution (pH close to 2.5), which must provide selective coloration of intestinal mucus, cartilage and mast cells, but not of nuclei. After alcian blue staining and treatment with 0.03 M Na2CO3 or Li2CO3 to convert the bound dye to a pigment, the Feulgen stain for DNA is applied. Dyes to be certified as alcian blue 8G or the equivalent must resist extraction by the 5 M HCl used in the Feulgen reaction. Dyes to be certified as alcian blue variant are not required to be convertible to acid-insoluble pigments, but they must dissolve easily in water at pH 5.7 containing 0.5 M magnesium chloride and the dye must remain in solution for at least 24 h. A critical electrolyte concentration (CEC) staining test also is described; this must be passed for certification of an alcian blue variant. Successful CEC staining is also a desirable property of alcian blue 8G or equivalent, but not essential for certification of an otherwise satisfactory batch. The spectrophotometric criteria for alcian blue dyes also are revised; a wider range of absorption maximum (605-634 nm) is allowed. The dye powders used in published staining techniques with the original alcian blue 8G were 40-60% dye, but some modern alcian blue dyes have dye content as high as 90%. The BSC's assay for dye content is not a criterion for certification, but it should influence the amount of dye to include in a staining solution.

A review of curcumin as a biological stain and as a self-visualizing pharmaceutical agent.

Curcumin has been widely used to color textiles but, unlike other natural dyes such as hematoxylin or saffron, it rarely has been discussed as a biological stain. Aspects of the physicochemistry of curcumin relevant to biological staining and self-visualization, i.e., its acidic properties, lipophilicity, metal and pseudometal complexes, and optical properties, are summarized briefly here. Reports of staining of non-living biological specimens in sections and smears, both fixed and unfixed, including specimens embedded in resin, are summarized here. Staining of amyloid, boron and chromatin are outlined and possible reaction mechanisms discussed. Use of curcumin as a vital stain also is described, both in cultured monolayers and in whole organisms. Staining mechanisms are considered especially for the selective uptake of curcumin into cancer cells. Staining with curcumin labeled nanoparticles is discussed. Toxicity and safety issues associated with the dye also are presented.

Uptake and localization mechanisms of fluorescent and colored lipid probes. Part 3. Protocols for predicting intracellular localization of lipid probes using QSAR models.

We provide detailed protocols for applying the QSAR decision-rule models described in Part 2 of this paper. These procedures permit prediction of the intracellular localization of fluorescent probes or of any small molecular xenobiotic whether fluorescent or not. Also included is a set of notes that give practical advice on various possible problems and limitations of the methods, together with a flow chart that provides a graphical algorithmic summary of the QSAR models.

Uptake and localization mechanisms of fluorescent and colored lipid probes. Part 2. QSAR models that predict localization of fluorescent probes used to identify ("specifically stain") various biomembranes and membranous organelles.

We discuss a variety of biological targets including generic biomembranes and the membranes of the endoplasmic reticulum, endosomes/lysosomes, Golgi body, mitochondria (outer and inner membranes) and the plasma membrane of usual fluidity. For each target, we discuss the access of probes to the target membrane, probe uptake into the membrane and the mechanism of selectivity of the probe uptake. A statement of the QSAR decision rule that describes the required physicochemical features of probes that enable selective staining also is provided, followed by comments on exceptions and limits. Examples of probes typically used to demonstrate each target structure are noted and decision rule tabulations are provided for probes that localize in particular targets; these tabulations show distribution of probes in the conceptual space defined by the relevant structure parameters ("parameter space"). Some general implications and limitations of the QSAR models for probe targeting are discussed including the roles of certain cell and protocol factors that play significant roles in lipid staining. A case example illustrates the predictive ability of QSAR models. Key limiting values of the head group hydrophilicity parameter associated with membrane-probe interactions are discussed in an appendix.

News from the Biological Stain Commission no. 15.

In the 15(th) issue of News from the Biological Stain Commission (BSC), under the heading of Regulatory affairs, the Biological Stain Commission's International Affairs Committee presents information from the plenary meetings of the International Standards Organization ISO/TC 212 Clinical laboratory testing and in vitro diagnostic test systems held on August 22-24, 2012 in Berlin, Germany. An additional discussion of the use of food dyes in India also is included.

Predicting small molecule fluorescent probe localization in living cells using QSAR modeling. 1. Overview and models for probes of structure, properties and function in single cells.

Small molecule fluorochromes (synonyms: biosensors, chemosensors, fluorescent probes, vital stains) are widely used to investigate the structure, composition, physicochemical properties and biological functions of living cells, tissues and organisms. Selective entry and accumulation within particular cells and cellular structures are key processes for achieving these diverse objectives. Despite the complexities, probes routinely are applied using standard protocols, often without experimenter awareness of what factors that control accumulation and localization. The mechanisms of many such selective accumulations, however, now are known. Moreover, the influence of physicochemical properties of probes on their uptake and localization often can be defined numerically, hence predicted, using quantitative structure activity relations (QSAR) models with its required numerical structure parameters (or "descriptors"). The state of the art of this approach is described. Available QSAR models are summarized for uptake into cells and localization in the cytosol, endoplasmic reticulum, generic biomembranes, Golgi apparatus, lipid droplets, lysosomes/endosomes, mitochondria, eukaryotic nuclei (histones and DNA), plasma membrane, and ribosomal RNA (cytoplasmic and nucleolar). Integration of such core models to both aid understanding and troubleshooting of current fluorescent probes and to assist the design of novel probes is outlined and illustrated using case examples. Limitations and generic problems arising with this approach and comments on application of such approaches to xenobiotics other than probes, e.g., drugs and herbicides, together with a brief note about an alternative approach to prediction, are given.

Predicting small molecule fluorescent probe localization in living cells using QSAR modeling. 2. Specifying probe, protocol and cell factors; selecting QSAR models; predicting entry and localization.

We describe the practical issues and the methodological procedures that must be carried out to construct and use QSAR models for predicting localization of probes in single cells. We address first the determination of probe factors starting with a consideration of the chemical nature of probe molecules present. What is their identity? Do new compounds arise in incubation media or intracellularly? For each probe, how many distinct chemical species are present? For each probe species, the derivation of the following numerical structure parameters, or descriptors, is set out with worked examples of electric charge and acid/base strength (Z and pKa); hydrophilicity/lipophilicity (log P); amphiphilicity (AI and HGH); conjugated bond number and largest conjugated fragment (CBN and LCF); width and length (W and L); and molecular and ionic weights, head group size and substituent bulk (MW, IW, HGS and SB). Next, protocol factors are specified by focusing separately on the mode of introduction of the probe to the cells, other application phenomena, and factors that influence directly observations of outcomes. Cell factors then are specified by considering separately structural and functional aspects. The next step is to select appropriate QSAR models and to integrate probe, protocol and cell factors to predict the interactions of the probe with the cell. Finally, we use an extended case example to explore the intracellular localization of certain photodynamic therapy dyes to illustrate these procedures.

Predicting and avoiding subcellular compartmentalization artifacts arising from acetoxymethyl ester calcium imaging probes. The case of fluo-3 AM and a general account of the phenomenon including a problem avoidance chart.

Stimulated by difficulties experienced when using fluo-3 AM, we developed a general mechanistic model to aid understanding and practical application of calcium probes applied as acetoxymethyl (AM) esters. Several practical issues previously overlooked or under-emphasized are considered by this model. First, some AM ester probes are "super" lipophilic, e.g., calcium orange, fluo-3, fura red, and these are trapped in the plasma membrane. Entry of such compounds into cells requires the presence of serum albumin in the incubation medium or esterase in the plasma membrane or both. Second, visible cytosolic calcium signals require significant cytosolic esterase, which varies considerably among cell lines and within cell populations of a single cell line. Finally, compartmentalization artefacts are most likely when incompletely hydrolyzed esters are present in the cytosol. This can occur because of low cytosolic esterase concentration or activity, and especially when long incubation times or high extracellular probe concentrations are used. An additional factor favoring compartmentalization is the presence of the "salt" form of the probe in the cytosol in the absence of significant concentrations of calcium ions. We provide an algorithmic chart to aid assessment of possible compartmentalization, guides to relevant QSAR models, and notes on estimation of the structural parameters required when using these models.

News from the Biological Stain Commission No. 11.

The 11th issue of News from the Biological Stain Commission (BSC) provides our first impressions of the REACH and ECHA programs. We intend to give a more thorough account of what these important programs actually mean in later editions of News from the Biological Stain Commission. Under the heading of Regulatory Affairs, the Biological Stain Commission's International Affairs Committee presents information from the opening session of the meeting of the International Standards Organization ISO/TC 212 Clinical laboratory testing and in vitro diagnostic test systems held on 2-4 June 2010 in Seoul, Republic of Korea.

Report on the 2nd Workshop on Fluorescence Chemosensors and Bioimaging, Dalian, China: one participant's view.

I was invited to this Workshop, because I have published papers on the mechanisms of action of small molecule fluorescent probes used with living cells. The Workshop provided an opportunity to interact with some significant figures in the chemosensor and bioimaging field from across the planet; to spend time with a large, friendly and active group of local investigators and their graduate students; and to take a brief look at a vibrant modern city. Many scientific connections were made and collaborations planned for the Biological Stain Commission and for my own future work.

Tracking living decapod larvae: mass staining of eggs with neutral red prior to hatching.

Mass staining of decapod females carrying eggs, with subsequent identification of hatched larvae in the environment, is a research tool with great potential for field ecologists wishing to track the movements of larvae. For this to be achieved, however, numerous requirements must be met. These include adequate dye solubility, short staining time, dye penetration through different tissues, dye retention within the organism, absence of toxic and behavioral effects, low visibility to predators of stained larvae, no loss of staining owing to preservatives and low cost. The dye, neutral red, appears to meet most of these requirements. This dye was used in aliquots of 0.7 g/770 ml seawater applied to the females of Norway lobster (Nephrops norvegicus) and European lobster (Homarus gammarus) for 10 min. This procedure stained lobster eggs and embryos so that hatched larvae could be distinguished easily by fluorescence microscopy from larvae that hatched from unstained eggs. Stained larvae that were preserved in 4% formaldehyde in seawater were still stained after 1 year. Larvae should not come in contact with ethanol, because it extracts the dye rapidly.

Biomedical applications and chemical nature of three dyes first synthesized by Raphael Meldola: isamine blue, Meldola's blue and naphthol green B.

Brief accounts are given of the chemical nature, and past and current biomedical applications of three dyes first synthesized by Raphael Meldola: isamine blue, Meldola's blue and naphthol green B.

How Romanowsky stains work and why they remain valuable - including a proposed universal Romanowsky staining mechanism and a rational troubleshooting scheme.

An introduction to the nomenclature and concept of "Romanowsky stains" is followed by a brief account of the dyes involved and especially the crucial role of azure B and of the impurity of most commercial dye lots. Technical features of standardized and traditional Romanowsky stains are outlined, e.g., number and ratio of the acidic and basic dyes used, solvent effects, staining times, and fixation effects. The peculiar advantages of Romanowsky staining are noted, namely, the polychromasia achieved in a technically simple manner with the potential for stain intensification of "the color purple." Accounts are provided of a variety of physicochemically relevant topics, namely, acidic and basic dyeing, peculiarities of acidic and basic dye mixtures, consequences of differential staining rates of different cell and tissue components and of different dyes, the chemical significance of "the color purple," the substrate selectivity for purple color formation and its intensification in situ due to a template effect, effects of resin embedding and prior fixation. Based on these physicochemical phenomena, mechanisms for the various Romanowsky staining applications are outlined including for blood, marrow and cytological smears; G-bands of chromosomes; microorganisms and other single-cell entities; and paraffin and resin tissue sections. The common factors involved in these specific mechanisms are pulled together to generate a "universal" generic mechanism for these stains. Certain generic problems of Romanowsky stains are discussed including the instability of solutions of acidic dye-basic dye mixtures, the inherent heterogeneity of polychrome methylene blue, and the resulting problems of standardization. Finally, a rational trouble-shooting scheme is appended.

Identifying apoptotic cells with the 3-hydroxyflavone derivative F2N12S, a ratiometric fluorescent small molecule probe selective for plasma membranes: a possible general mechanism for selective uptake into apoptotic cells.

The mechanism of selective targeting of the plasma membrane of apoptotic cells by F2N12S, a recently reported ratiometric, fluorescent small molecule probe, was analyzed using decision-rule QSAR models. Selectivity was determined by a combination of the probe's weak amphiphilicity and slow flip-flop with the increased plasma membrane fluidity of apoptotic cells. The probable chemical features required for such probes may be defined in terms of numerical structural parameters as: 3.5 < AI < ∼ 5.5; log P < 5.0; HGS > 400 (where AI, log P and HGS parameters model amphiphilicity, lipophilicity and headgroup size, respectively). When HGS is <400, compounds are initially membrane selective, but subsequently are internalized.

Uptake and localization mechanisms of fluorescent and colored lipid probes. 1. Physicochemistry of probe uptake and localization, and the use of QSAR models for selectivity prediction.

We outline the factors involved in precise targeting of lipids and membranes by probes, namely, lipid and probe chemistry, geometry/topography of probe delivery, and probe uptake kinetics. The special case of probe orientation within membranes also is considered. The varieties of commercially available fluorophores are described, and an overview of probe physicochemical properties (amphiphilicity, conjugated system size, electrical properties, head group size, lipophilicity and solubility) is provided together with notes on their parameterization. Probe-lipid physicochemical interactions, and their relations to parameterization, then are discussed including the nature and derivation of decision-rule QSAR models, partitioning and insertion of probes into bulk lipids and complications of this, partitioning and insertion of probes into membranes, and flip-flop of probes across membrane leaflets. A general QSAR algorithm for understanding lipid probe application then is set out. Problems and limitations are outlined. Biological issues include varied biomembrane composition, cell line effects and toxicity of fluorescent probes. Methodological issues include difficulties of estimating certain numerical structure parameters, the impure character of many fluorochromes and dyes, and the perturbation of biomembrane structure by fluorescent probes.

Real-time imaging of bacteria in living mice using a fluorescent dye.

A novel technique developed in the laboratories of Bradley D. Smith and David Piwnica-Worms for imaging bacterial infections in intact living nude mice using a novel fluorescent dye, a conjugate of a NIR carbocyanine dye and two zinc(II) dipicolylamine units, allows relatively deep imaging of bacterial infection in real time. The behavior of the mice indicated good tolerance of the probe. The probe's water-octanol partition coefficient calculated by Hansch and Leo's procedure demonstrates that it is slightly lipophilic and therefore could enter mouse cells. Extant values of the physicochemical and spectroscopic parameters relevant to practical use are tabulated.

Binding of cationic dyes to DNA: distinguishing intercalation and groove binding mechanisms using simple experimental and numerical models.

Simple methods for predicting intercalation or groove binding of dyes and analogous compounds with double stranded DNA are described. The methods are based on a quantitative assessment of the aspect (width to length) ratio of the dyes. The procedures were validated using a set of 38 cationic dyes of varied chemical structures binding to well oriented DNA fibers and assessing binding orientation by linear dichroism and polarized fluorescence. We demonstrated that low aspect ratio dyes bound by intercalation, whereas more rod-like dyes were groove binders. Some problems that result and possible applications are discussed briefly.

Selective labeling of lipid droplets in aldehyde fixed cell monolayers by lipophilic fluorochromes.

We evaluated a number of lipophilic dyes and fluorochromes, including oxazone and thiazone derivatives of oxazine and thiazine dyes, scintillator agents, a carotenoid and a metal-porphyrin complex for visualization of lipid droplets within aldehyde fixed cultured HeLa and BGC-1 cells. Observation under ultraviolet, blue or green exciting light revealed selective fluorescence of lipid droplets, particularly after treatment with aqueous solutions of Nile blue and brilliant cresyl blue oxazones, toluidine blue thiazone, or propylene glycol solutions of canthaxanthin, ethyl-BAO, and ZnTPyP. Mounting in water was required to maintain the fluorescence of lipids; the use of glycerol, Mowiol or Vectashield was not adequate. The effect of dye structure on staining intensity was assessed with the aid of numerical structure parameters modeling lipophilicity (HI and log P), overall size (MW) and the size of the conjugated system (conjugated bond number; CBN). The best stains for lipid droplets were relatively lipophilic (HI > 4.0, log P > 5.0), of small size overall (MW < 370), with small conjugated systems (CBN < 24), and not significantly amphiphilic. The two hydrophobic-hydrophilic parameters (the classic log P and the hydrophobic index, HI; values calculated by molecular modeling software) were highly correlated; however, HI was a more suitable hydrophobicity index for the dyes studied here.