Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo .^1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:

1. stem and progenitor cell quiescence, proliferation and/or differentiation^6-8
2. antigen-driven membrane transfer⁹ and/or precursor cell proliferation^3,4,10-18 and
3. immune regulatory and effector cell function^1,18-21.

Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes^1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins^4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class^2-4,16,18,24.The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:

1. Failure to achieve bright, uniform, reproducible labeling . This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.
2. Suboptimal fluorochrome combinations and/or failure to include critical compensation controls . Tracking dye fluorescence is typically 10² - 10³ times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.
3. Failure to obtain a good fit with peak modeling software . Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.

Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.     

Synthesis of Novel Tyrosinyl FRET Cassettes,Terminators, and Their Potential Use in DNA Sequencing

Abstract

Fluorescence resonance energy transfer (FRET) dye labeled cassettes and terminators with one or more donor dyes (fluorescein) and acceptor dye (rhodamine dyes) with benzofuran or tyrosine linker moieties were synthesized. These terminators were evaluated for their energy transfer and DNA sequencing potential using thermostable DNA polymerase.     

Imaging Brain Activity With Voltage- and Calcium-Sensitive Dyes

This paper presents three examples of imaging brain activity with voltage- or calcium-sensitive dyes and then discusses the methodological aspects of the measurements that are needed to achieve an optimal signal-to-noise ratio.Internally injected voltage-sensitive dye can be used to monitor membrane potential in the dendrites of invertebrate and vertebrate neurons in in vitro preparations.Both invertebrate and vertebrate ganglia can be bathed in voltage-sensitive dyes to stain all of the cell bodies in the preparation. These dyes can then be used to follow the spike activity of many neurons simultaneously while the preparations are generating behaviors.Calcium-sensitive dyes that are internalized into olfactory receptor neurons in the nose will, after several days, be transported to the nerve terminals of these cells in the olfactory bulb. There they can be used to measure the input from the nose to the bulb.Three kinds of noise are discussed. a. Shot noise from the random emission of photons from the preparation. b. Vibrational noise from external sources. c. Noise that occurs in the absence of light, the dark noise.Three different parts of the light measuring apparatus are discussed: the light sources, the optics, and the cameras.The major effort presently underway to improve the usefulness of optical recordings of brain activity are to find methods for staining individual cell types in the brain. Most of these efforts center around fluorescent protein sensors of activity.     

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis^1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation^1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals^3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane⁹. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal¹⁰, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable¹¹.Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)^12,13.     

Quirks of dye nomenclature. 5. Rhodamines

Rhodamines were first produced in the late 19^th century, when they constituted a new class of synthetic dyes. These compounds since have been used to color many things including cosmetics, inks, textiles, and in some countries, food products. Certain rhodamine dyes also have been used to stain biological specimens and currently are widely used as fluorescent probes for mitochondria in living cells. The early history and current biological applications are sketched briefly and an account of the ambiguities, complications and confusions concerning dye identification and nomenclature are discussed.     

Efficient,Cyanine Dye Based Bilayer Solar Cells

Simple bilayer solar cells, using commercially available cationic cyanine dyes as donors and evaporated C₆₀ layer as an acceptor are prepared. Cyanine dyes with absorption maxima of 578, 615 and 697 nm having either perchlorate or hexafluorophosphate counter‐ions are evaluated. The perchlorate dye leads to cells with S‐shape current‐voltage curves; only the dyes with the hexafluorophosphate counter‐ions lead to efficient solar cells. When the wide bandgap dyes are employed, S‐shape current‐voltage curves are obtained when the conductive polymer PEDOT:PSS is used as hole transport layer. Substitution of PEDOT:PSS with MoO₃ leads to cells with more rectangular current–voltage curves and high fill factors. Additionally, the cells using the MoO₃ layer for hole extraction lead to high open circuit voltages of 0.9 V. In the case that a low bandgap hexafluorophosphate dye is used with the HOMO above that of the PEDOT:PSS the cell performance is independent on the type of hole transport layer employed. Using this approach, bilayer solar cells are obtained with power efficiencies ranging from 1.8 to 2.9% depending on the particular dye employed. These are impressive numbers for bilayer solar cell that are partially solution processed in ambient conditions.     

Surfactant modified barley straw for removal of acid and reactive dyes from aqueous solution

A barley straw was modified by a surfactant, cetylpyridinium chloride, and used as an adsorbent for acid (acid blue 40) and reactive dye (reactive black 5) adsorption in aqueous solution. Characterization of the modified barley straw was performed using N₂ adsorption, titration, and FT-IR analysis. It was found that the surfactant modified barley straw exhibits higher adsorption to acid blue 40 than reactive black 5 and adsorption of the dyes is influenced by several parameters such as dye initial concentration, adsorbent dosage, solution pH, and adsorption temperature. Adsorption isotherms show that maximum adsorption of acid blue 40 and reactive black 5 is 1.02 × 10⁻⁴ and 2.54 × 10⁻⁵ mol/g, respectively. Desorption studies show that both dyes are strongly bounded with the adsorbent and exhibit low desorption.     

Dyes,trypanosomiasis and DNA: a historical and critical review

Abstract

Trypanosomiasis, a group of diseases including sleeping sickness in humans and Nagana in cattle in Africa, and Chagas’ disease in South America, remains a considerable problem in the 21^st century. The therapies that are available, however, usually have their roots in the “dye therapy” of a century ago, knowledge gained at the microscope from parasite staining procedures and converted to chemotherapy based on compounds closely related to the laboratory reagents. Dyes such as trypan red and trypan blue led to the development of suramin, while cationic nitrogen heterocyclic dyes furnished examples of the phenanthridinium class, such as ethidium (homidium) and isometamidium. Both suramin and isometamidium remain in use. Owing to mutagenicity issues, the presence of ethidium among the phenanthridinium dyes has led to concerns over the clinical use of related derivatives. There are several mechanisms for dye-DNA interaction, however, including possible hydrogen bonding of dye to the polymer, and these are discussed together with structure-activity relations and cellular localization of the phenanthridine and isomeric acridines involved. Better understanding of nucleic acid binding properties has allowed the preparation of more effective phenanthridinium analogues intended for use as anticancer/antiviral therapy.     

Evidence on manganese peroxidase and tyrosinase expression during decolorization of textile industry dyes by Trichosporon akiyoshidainum

Textile dyes are engineered to be resistant to environmental conditions. During recent years the treatment of textile dye effluents has been the focus of significant research because of the potentially low cost of the process. Mechanisms of biological textile dye decolorization depend greatly on the chemical structure of the dye and the microorganisms used. While basidiomycetous filamentous fungi are well recognized for dye decolorization through ligninolytic enzymes, reports on textile dye decolorization mechanisms of basidiomycetous yeasts have been scarce. Decolorization of several textile dyes by Trichosporon akiyoshidainum occurs during the first 12 h of cultivation. This fast decolorization process could not be solely related to siderophore production or dye sorption to biomass; it was shown to be a co-metabolic process. T. akiyoshidainum could use glucose, sucrose, and maltose as alternative carbon sources, and urea as an alternative nitrogen source with similar decolorization rates. The activity of two enzymes, manganese peroxidase and tyrosinase, were induced by the presence of dyes in the culture media, pointing to their potential role during the decolorization process. Manganese peroxidase titers reached 666 U l⁻¹ to 10538 U l⁻¹, while tyrosinase titers ranged between 84 U l⁻¹ and 786 U l⁻¹, depending on the dye tested. The present work provides a useful background to propose new eco-friendly alternatives for wastewater treatment in textile dying industries.     

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.     

Synthesis and DNA binding profile of monomeric,dimeric, and trimeric derivatives of crystal violet

Monomeric, dimeric, and trimeric derivatives of the triphenylmethane dye crystal violet (1a–1f) have been synthesized for the purpose of evaluating their affinity and sequence selectivity for duplex DNA. Competitive ethidum displacement assays indicate that 1a–1f have apparent association constants for CT DNA in the range of 1.80–16.2 × 10⁷ M⁻¹ and binding site sizes of 10–14 bp. Viscosity experiments performed on ligand 1f confirmed that these dyes associate with duplex DNA by a non-intercalative mode of binding. Circular dichroism and competition binding studies of the tightest binding ligand 1e with known major and minor groove binding molecules suggest that these dye derivatives likely occupy the major groove of DNA. Data from the binding of 1e to polynucleotides indicate close to an order of magnitude preference for associating with AT rich homopolymers over GC rich homopolymers, suggesting a shape-selective match of the sterically bulky ligand with DNA containing a wider major groove.     

Charge translocation by the Na,K-pump: I. Kinetics of local field changes studied by time-resolved fluorescence measurements

Summary Membrane fragments containing a high density of Na, K-ATPase can be noncovalently labeled with amphiphilic styryl dyes (e.g., RH 421). Phosphorylation of the Na,K-ATPase by ATP in the presence of Na⁺ and in the absence of K⁺ leads to a large increase of the fluorescence of RH 421 (up to 100%). In this paper evidence is presented that the styryl dye mainly responds to changes of the electric field strength in the membrane, resulting from charge movements during the pumping cycle: (i) The spectral characteristic of the ATP-induced dye response essentially agrees with the predictions for an electrochromic shift of the absorption peak. (ii) Adsorption of lipophilic anions to Na, K-ATPase membranes leads to an increase, adsorption of lipophilic cations to the decrease of dye fluorescence. These ions are known to bind to the hydrophobic interior of the membrane and to change the electric field strength in the boundary layer close to the interface. (iii) The fluorescence change that is normally observed upon phosphorylation by ATP is abolished at high concentrations of lipophilic ions. Lipophilic ions are thought to redistribute between the adsorption sites and water and to neutralize in this way the change of field strength caused by ion translocation in the pump protein. (iv) Changes of the fluorescence of RH 421 correlate with known electrogenic transitions in the pumping cycle, whereas transitions that are known to be electrically silent do not lead to fluorescence changes. The information obtained from experiments with amphiphilic styryl dyes is complementary to the results of electrophysiological investigations in which pump currents are measured as a function of transmembrane voltage. In particular, electrochromic dyes can be used for studying electrogenic processes in microsomal membrane preparations which are not amenable to electrophysiological techniques.Deceased (September 13, 1990).     

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.     

Combining Voltage and Calcium Imaging from Neuronal Dendrites

The ability to monitor membrane potential (V _m) and calcium (Ca²⁺) transients at multiple locations on the same neuron can facilitate further progress in our understanding of neuronal function. Here we describe a method to combine V _m and Ca²⁺ imaging using styryl voltage sensitive dyes and Fura type UV-excitable Ca²⁺ indicators. In all cases V _m optical signals are linear with membrane potential changes, but the calibration of optical signals on an absolute scale is presently possible only in some neurons. The interpretation of Ca²⁺ optical signals depends on the indicator Ca²⁺ buffering capacity relative to the cell endogenous buffering capacity. In hippocampal CA1 pyramidal neurons, loaded with JPW-3028 and 300 μM Bis-Fura-2, V _m optical signals cannot be calibrated and the physiological Ca²⁺ dynamics are compromised by the presence of the indicator. Nevertheless, at each individual site, relative changes in V _m and Ca²⁺ fluorescence signals under different conditions can provide meaningful new information on local dendritic integration. In cerebellar Purkinje neurons, loaded with JPW-1114 and 1 mM Fura-FF, V _m optical signals can be calibrated in terms of mV and Ca²⁺ optical signals quantitatively reveal the physiological changes in free Ca²⁺. Using these two examples, the method is explained in detail.     

Decolorization kinetics of the azo dye Reactive Red 2 under methanogenic conditions: effect of long-term culture acclimation

The biological decolorization of the textile azo dye Reactive Red 2 was investigated using a mixed, mesophilic methanogenic culture, which was developed with mixed liquor obtained from a mesophilic, municipal anaerobic digester and enriched by feeding a mixture of dextrin/peptone as well as media containing salts, trace metals and vitamins. Batch decolorization assays were conducted with the unacclimated methanogenic culture and dye decolorization kinetics were determined as a function of initial dye, biomass, and carbon source concentrations. Dye decolorization was inhibited at initial dye concentrations higher than 100 mg l^-1 and decolorization kinetics were described based on the Haldane model. The effect of long-term culture exposure to the reactive dye on decolorization kinetics, culture acclimation, as well as possible dye mineralization was tested using two reactors fed weekly for two years with an initial dye concentration of 300 mg l^-1 and a mixture of dextrin/peptone. The maximum dye decolorization rate after a 2-year acclimation at an initial dye concentration of 300 mg l^-1 was more than 10-fold higher as compared to that obtained with the unacclimated culture. Aniline and the o-aminohydroxynaphthalene derivative resulting from the reductive azo bond cleavage of the dye were detected, but further transformation(s) leading to dye mineralization were not observed. Reactive Red 2 did not serve as the carbon and energy source for the mixed culture, and dye decolorization was sustained by the continuous addition of dextrin and peptone. Thus, biological decolorization of reactive azo dyes is feasible under conditions of low redox potential created and maintained in overall methanogenic systems, but supply of a biodegradable carbon source is necessary.     

Dye decolorization by sub-tropical basidiomycetous fungi and the effect of metals on decolorizing ability

White-rot basidiomycetous fungi from sub-tropical forests plus a Phanerochaete chrysosporium control were able to decolorize several azo, triphenylmethane and heterocyclic/polymeric dyes over 14 days. The effects of metal ions on decolorizing ability towards the dye Poly-R varied. Two sub-tropical strains were capable of decolorization in the presence of up to 0.25 mM Cd²⁺, Cu²⁺ and Zn²⁺, whereas decolorization by P. chrysosporium was completely inhibited by all metals at concentrations as low as 0.1 mM. In all cases decolorizing ability was more sensitive than biomass production to metal inhibition.     

Decolorization of diazo dye Direct Red 81 by a novel bacterial consortium

Summary Samples collected from various effluent-contaminated soils in the vicinities of dyestuff manufacturing units of Ahmedabad, India, were studied for screening and isolation of organisms capable of decolorizing textile dyes. A novel bacterial consortium was selected on the basis of rapid decolorization of Direct Red 81 (DR 81), which was used as model dye. The bacterial consortium exhibited 90% decolorization ability within 35 h. Maximum rate of decolorization was observed when starch (0.6 g l⁻¹) and casein (0.9 g l⁻¹) were supplemented in the medium. Decolorization of DR 81 was monitored by high performance thin layer chromatography, which indicated that dye decolorization was due to its degradation into unidentified intermediates. The optimum dye-decolorizing activity of the culture was observed at pH 7.0 and incubation temperature of 37 °C. Maximum dye-decolorizing efficiency was observed at 200 mg l⁻¹ concentration of DR 81. The bacterial consortium had an ability to decolorize nine other structurally different azo dyes.     

Behavioural and morphological evidence for the involvement of glial cell activation in delta opioid receptor function: implications for the development of opioid tolerance

The molecular identity and pharmacological properties of mechanically gated ion channels in sensory neurons are poorly understood. We show that FM1-43, a styryl dye used to fluorescently label cell membranes, permeates mechanosensitive ion channels in cultured dorsal root ganglion neurons, resulting in blockade of three previously defined subtypes of mechanically activated currents. Blockade and dye uptake is voltage dependent and regulated by external Ca²⁺. The structurally related larger dye FM3-25 inhibited mechanically activated currents to a lesser degree and did not permeate the channels. In vivo, FMI-43 decreases pain sensitivity in the Randall-Selitto test and increases the withdrawal threshold from von Frey hairs, together suggesting that the channels expressed at the cell body in culture mediate mechanosensation in the intact animal. These data give further insight into the mechanosensitive ion channels expressed by somatosensory neurons and suggest FM dyes are an interesting tool for studying them.     

Transforming Benzophenoxazine Laser Dyes into Chromophores for Dye‐Sensitized Solar Cells: A Molecular Engineering Approach

The refunctionalization of a series of four well‐known industrial laser dyes, based on benzophenoxazine, is explored with the prospect of molecularly engineering new chromophores for dye‐sensitized solar cell (DSC) applications. Such engineering is important since a lack of suitable dyes is stifling the progress of DSC technology. The conceptual idea involves making laser dyes DSC‐active by chemical modification, while maintaining their key property attributes that are attractive to DSC applications. This molecular engineering follows a stepwise approach. First, molecular structures and optical absorption properties are determined for the parent laser dyes: Cresyl Violet ( 1 ), Oxazine 170 ( 2 ), Nile Blue A ( 3 ), Oxazine 750 ( 4 ). These reveal structure‐property relationships which define the prerequisites for computational molecular design of DSC dyes; the nature of their molecular architecture (D‐π‐A) and intramolecular charge transfer. Second, new DSC dyes are computationally designed by the in silico addition of a carboxylic acid anchor at various chemical substitution points in the parent laser dyes. A comparison of the resulting frontier molecular orbital energy levels with the conduction band edge of a TiO₂ DSC photoanode and the redox potential of two electrolyte options I^?/I₃^? and Co^(II/III)tris(bipyridyl) suggests promise for these computationally designed dyes as co‐sensitizers for DSC applications.     

Interpretation and Optimization of Absorbance and Fluorescence Signals from Voltage-sensitive Dyes

Voltage-sensitive dyes produce absorbance and fluorescence changes that can be used to image voltage. The present study develops a systematic approach to the optimization of these signals. A mathematical analysis assesses the dye optical density (OD) that optimizes the signal-to-noise ratio in absorbance and fluorescence measurements. The signal-to-noise ratio is maximal for a dye OD of 2 (natural logarithm) in absorbance and ~1 in fluorescence. The fluorescence result is approximate because, in contrast to absorbance, the optimal dye OD varies with the amount of scattering and intrinsic absorbance of the tissue. The signal-to-noise ratio of absorbance is higher in thick preparations such as brain slices; fluorescence is superior in thin preparations such as cell culture. The optimal OD for absorbance and fluorescence, as well as the superiority of absorbance, were confirmed experimentally on hippocampal slices. This analysis also provided insight into the interpretation of signals normalized to resting light intensities. With both absorbance and fluorescence, the normalized signal (I/I) varies with OD, and does not reflect the change in dye absorbance. In absorbance this problem is remedied by dividing I/I by the dye OD to obtain the absorbance change. For fluorescence a correction is possible, but is more complicated. Because this analysis indicates that high levels of stain optimize the signal-to-noise, dyes were tested for pharmacological actions and phototoxicity. The absorbance dye RH155 was found to have pharmacological action at high staining levels. The fluorescent dye RH414 was phototoxic. Adverse effects could not be detected with the absorbance dye RH482.