On-line cell mass monitoring of Saccharomyces cerevisiae cultivations by multi-wavelength fluorescence

The catalyst in bioprocesses, i.e. the cell mass, is one of the most challenging and important variables to monitor in bioprocesses. In the present study, cell mass in cultivations with Saccharomyces cerevisiae was monitored on-line with a non-invasive in situ placed sensor measuring multi-wavelength culture fluorescence. The excitation wavelength ranged from 270 to 550 nm with 20 nm steps and the emission wavelength range was from 310 to 590 nm also with 20 nm steps. The obtained spectra were analysed chemometrically with the multi-way technique, parallel factor analysis (PARAFAC), resulting in a decomposition of the multivariate fluorescent landscape, whereby underlying spectra of the individual intrinsic fluorophors present in the cell mass were estimated. Furthermore, gravimetrically determined cell mass concentration was used together with the fluorescence spectra for calibration and validation of multivariate partial least squares (PLS) regression models. Both two- and three-way models were calculated, the models behaved similarly giving root mean square error of prediction (RMSEPs) of 0.20 and 0.19 g l(-1), respectively, when test set validation was used. Based on this work, it is evident that on-line monitoring of culture fluorescence can be used for estimation of the cell mass concentration during cultivations.     

On-line bioprocess monitoring with a multi-wavelength fluorescence sensor using multivariate calibration

Cultivations of Pseudomonas fluorescens were monitored with a multi-wavelength on-line fluorescence sensor. The multi-wavelength fluorometer used excitation light from 270 to 550 nm with 20 nm steps and measured fluorescence emission from 310 to 590 nm. The fluorescence, on-line exhaust gas measurements and off-line analysis of nitrate, succinate, optical density and protein were compared chemometrically by multivariate calibration, i.e. computing partial least square (PLS) regression models. Based on the multivariate regression models, it was possible to determine CO2 and O2 composition in the exhaust gas (the correlation coefficients, R2 between the predicted values by the PLS model and the measured values was 0.97 for CO(2) and 0.97 for O2, respectively). Also to make quantitative determinations of succinate (R(2) = 0.97), protein (R(2) = 0.94), optical density (R(2) = 1.0) and nitrate (R(2) = 0.98) in the medium based on the fluorescence spectra. Only a limited data set was available but the results indicated that the sensor could indirectly determine non-fluorescent compounds, i.e. nitrate and succinate, which probably is due to the stoichiometric relationship between fluorescent cellular components and non-fluorescent compounds. Consequently multi-wavelength fluorescence is an interesting technique for a wide range of applications.     

Real-time multi-wavelength fluorescence imaging of living cells

We describe a new real-time fluorescence video microscope design for capturing intensified images of cells containing dual wavelength "ratio" dyes or multiple dyes. The microscope will perform real-time capture of two separate fluorescence emission images simultaneously, improving the time resolution of spatial distribution of fluorescence to video frame rates (30 frames/s or faster). Each emission wavelength is imaged simultaneously by one of two cameras, then digitized, background corrected and appropriately combined at standard video frame rates to be stored at high resolution on tape or digital video disk for further off-line analysis. Use of low noise, high sensitivity image intensifiers, coupled to CCD cameras produce stable, high contrast images using ultra low light levels with little persistence or bloom. The design has no moving parts in its optical train, which overcomes a number of technical difficulties encountered in the present filter wheel designs for multiple imaging. Coupled to compatible image processing software utilizing PC-AT computers, the new design can be built for a significantly lower cost than many presently available commercial machines. The system is ideal for ratio imaging applications; the software can calculate the ratio of fluorescence intensities pixel by pixel and provide the information to generate false-color images of the intensity data as well as other calculations based on the two images. Thus, it provides a powerful, inexpensive tool for studying the real-time kinetics of changes in living cells. Examples are presented for the kinetics of rapidly changing intracellular calcium detected by the calcium indicator probe indo-1 and the redistribution kinetics of multiple vital dyes placed in cells undergoing cell fusion.     

Determination of cell concentration in a plant cell suspension using a fluorescence microplate reader

Microscopic counting of plant cells is a very tedious and time-consuming process and is therefore seldom used to evaluate plant cell number on a routine basis. This study describes a fast and simple method to evaluate cell concentration in a plant cell suspension using a fluorescence microplate reader. Eschscholtzia californica cells were fixed in a mix of methanol and acetic acid (3:1) and stained with a fluorescent DNA binding dye (Hoechst 33258). Readings were done in a fluorescence microplate reader at 360/465 nm. Specific binding of the dye to double-stranded DNA was significantly favored over unspecific binding when 1.0 M Tris buffer at pH 7.5 containing 1.0 M NaCl and 75 microg ml(-1) of Hoechst 33258 was used. Fluorescence readings must be done between 4 min and 12 min following the addition of the staining solution to the sample. The microplate counting method provides a convenient, rapid and sensitive procedure for determining the cell concentration in plant cell suspensions. The assay has a linear detection range from 0.2 x 10(6) cells to 10.0 x 10(6) cells per milliliter (actual concentration in the tested cell suspension). The time needed to perform the microplate counting was 10% of that needed for the microscopic enumeration. However, this microplate counting method can only be used on genetically stable cell lines and on asynchronous cell suspensions.     

Viewing dynamic assembly of molecular complexes by multi-wavelength single-molecule fluorescence

Complexes of macromolecules that transiently self-assemble, perform a particular function, and then dissociate are a recurring theme in biology. Such systems often have a large number of possible assembly/disassembly intermediates and complex, highly branched reaction pathways. Measuring the single-step kinetic parameters in these reactions would help to identify the functionally significant pathways. We have therefore constructed a novel single-molecule fluorescence microscope capable of efficiently detecting the colocalization of multiple components in a macromolecular complex when each component is labeled using a different color fluorescent dye. In this through-objective excitation, total internal reflection instrument, the dichroic mirror conventionally used to spectrally segregate the excitation and emission pathways was replaced with small broadband mirrors. This design spatially segregates the excitation and emission pathways and thereby permits efficient collection of the spectral range of emitted fluorescence when three or more dyes are used. In a test experiment with surface-immobilized single-stranded DNA molecules, we directly monitored the time course of a hybridization reaction with three different oligonucleotides, each labeled with a different color dye. The experiment reveals which of the possible reaction intermediates were traversed by each immobilized molecule, measures the hybridization rate constants for each oligonucleotide, and characterizes kinetic interdependences of the reaction steps.     

Determination of tobramycin in serum using liquid chromatography-tandem mass spectrometry and comparison with a fluorescence polarisation assay

We have developed a tandem mass spectrometry (LC-MS-MS) method for measuring tobramycin concentrations in serum samples and have compared it with a fluorescence polarisation immunoassay. After protein precipitation with acetonitrile supernatant was injected into the LC-MS-MS system. A C(18) cartridge (4x2 mm) was eluted with a step gradient of 20-100% methanol containing HFBA. The retention times were, tobramycin 1.05 min and sisomycin 1.05 min. The MRM transitions were: m/z 467.8>163 (tobramycin) and m/z 447.8>160 (sisomycin). The limit of quantification was 0.15 mg/l and the assay was linear up to 50 mg/l. Assay precision was <6% within and between batch.     

Determination of homocysteine thiolactone and homocysteine in cell cultures using high-performance liquid chromatography with fluorescence detection

A sensitive and simple method utilising fluorometric detection for the simultaneous routine monitoring of homocysteine thiolactone (HTL) and homocysteine (Hcy) in biological samples has been developed. Separation relies on isocratic ion-pairing and reversed-phase chromatography while the principle of the detection is that the lactone ring in HTL molecule is cleaved with an alkali to produce Hcy, which reacts with ortho-phthalaldehyde (OPA) in the absence of an added thiol reagent to form a stable fluorescent derivative. The method has a sensitivity of 200 fmol of HTL and 100 fmol for Hcy in the sample. The present method was applied to the determination of HTL and Hcy in Hep G2 cell.     

Determination of angiotensin I-converting enzyme activity in cell culture using fluorescence resonance energy transfer peptides

An assay using fluorescence resonance energy transfer peptides was developed to assess angiotensin I-converting enzyme (ACE) activity directly on the membrane of transfected Chinese hamster ovary cells (CHO) stably expressing the full-length somatic form of the enzyme. The advantage of the new method is the possibility of using selective substrates for the two active sites of the enzyme, namely Abz-FRK(Dnp)P-OH for somatic ACE, Abz-SDK(Dnp)P-OH for the N domain, and Abz-LFK(Dnp)-OH for the C domain. Hydrolysis of a peptide bond between the donor/acceptor pair (Abz/Dnp) generates detectable fluorescence, allowing quantitative measurement of the enzymatic activity. The kinetic parameter K(m) for the hydrolysis of the three substrates by ACE in this system was also determined and the values are comparable to those obtained using the purified enzyme in solution. The specificity of the activity was demonstrated by the complete inhibition of the hydrolysis by the ACE inhibitor lisinopril. Therefore, the results presented in this work show for the first time that determination of ACE activity directly on the surface of intact CHO cells is feasible and that the method is reliable and sensitive. In conclusion, we describe a methodology that may represent a new tool for the assessment of ACE activity which will open the possibility to study protein interactions in cells in culture.     

Determination of microphytobenthic biomass using pulse-amplitude modulated minimum fluorescence

Microphytobenthos are the major intertidal primary producers in many estuarine systems and assessment of their biomass is essential in ecological studies. Algal biomass can be assessed by the use of proxy measurements, e.g. chlorophyll a (Chl a), using a lengthy and destructive extraction procedure. Fluorescence is a non-destructive and in situ alternative, recently applied to microphytobenthos, using the minimum Fluorescence signal (Fo) as a measurement of Chl a. In this study, Fo after 15 min of dark adaptation (Fo ¹⁵) showed a significant positive correlation with Chl a (r?=?0.84, p?n?=?24). The relationship between Fo ¹⁵ and Chl a extracted from sediment samples of increasing sample depth (0.2?2?mm) became less significant as sediment depth increased. The strongest relationship between Fo ¹⁵ and Chl a, expressed as concentration (mg?m^?2) and content (mg?kg^?1), was found when all data were combined, but nearly comparable levels of association were found for in situ measurements using sediment cores up to 2.0?mm deep. Results suggested that full dark adaptation was not always achieved because a significant negative correlation was observed between Fv/Fm and ambient irradiance for seasonal data. In addition, Fo ¹⁵ showed a more significant correlation with Chl a than with Fm ¹⁵, suggesting some residual level of non-photochemical quenching after 15 min in the dark. Despite the incomplete dark adaptation, the highly significant relationships between Fo ¹⁵ and both Chl a concentration and content suggest that the use of Fo ¹⁵ is a suitable in situ proxy method of estimating algal biomass.     

Determination of octopine by pre-column fluorescence derivatization using benzoin

A sensitive fluorimetric method for the determination of octopine, a member of opine family, is presented. The method is based on the formation of a fluorescent derivative of octopine with benzoin and the separation by high performance liquid chromatography using a reversed-phase column (Kaseisorb LC ODS-300) within 20 min. The octopine derivative is completely separated from other guanidino compounds including arginine which is generally very high in marine invertebrates. This method gives higher sensitivity, 5 pmol minimum detection, and better reproducibility than the electrophoresis method and colorimetric method.     

Detection and quantification of triazine herbicides using algal cell fluorescence

Summary A continuous-flow system is described which, by measuring fluorescence of the unicellular alga Chlorella, is capable of measuring concentrations of the triazine herbicide, simazine, as low as 60nM (approx 12g l^-1) within 5 minutes. Further developments are suggested to achieve the desired detection limit of 0.5nM. The use of such an instrument in environmental analysis is discussed.     

Determination of plasma ziprasidone using liquid chromatography with fluorescence detection

A liquid chromatographic procedure was developed for the determination of a new antipsychotic agent ziprasidone in plasma using fluorescence detection. A one-step liquid-liquid extraction from 1 ml of alkalinized plasma containing an internal standard alpha-ergocryptine using methyl-t-butyl ether afforded a greater than 84% recovery of ziprasidone. Chromatography was performed using a reversed-phase trimethylsilyl bonded silica column with a mobile phase of 72:28 phosphate buffer:acetonitrile at a flow rate of 1.5 ml/min. Detection of the eluted peaks was observed using excitation and emission wavelengths of 320 and 410 nm, respectively. Chromatographic run time did not exceed 14 min with no interference from endogenous material. The calibration curve was linear over the concentration range of 0.5 to 200 ng/ml and the inter- and intra-assay imprecision (CV) was less than 10%. The lower limit of quantitation was assessed at 0.5 ng/ml. Specificity of the method is demonstrated by the lack of interference from a large number of commonly used drugs and their metabolites in clinical use. The utility of the method is exemplified with the presentation of clinical data from patients receiving ziprasidone.     

Determination of EETs using microbore liquid chromatography with fluorescence detection

Epoxyeicosatrienoic acids (EETs) are cytochrome P-450 metabolites of arachidonic acid involved in the regulation of vascular tone. The method of microbore column high-performance liquid chromatography with fluorescence detection was developed to determine 14,15-EET, 11, 12-EET, and the mixture of 8,9-EET and 5,6-EET. Tridecanoic acid (TA) was used as an internal standard. EETs were reacted with 2-(2, 3-naphthalimino)ethyl trifluoromethanesulfonate (NT) to form highly fluorescent derivatives. A C(18) microbore column and a water-acetonitrile mobile phase were used for separation. Samples were excited at 259 nm, and the fluorescence was detected at 395 nm. The overall recoveries were 88% for EETs and 40% for TA. EETs were detected in concentrations as low as 2 pg (signal-to-noise ratio = 3). The method was used to determine the EET production from endothelial cells (ECs). Bradykinin and methacholine (10(-6) M) stimulated an increase in the production of EETs by ECs two- and fivefold, respectively. This sensitive method may be used for determination of EETs at low concentrations normally detected in complex biological samples.     

Single cell fluorescence imaging using metal plasmon-coupled probe

This work constitutes the first fluorescent imaging of cells using metal plasmon-coupled probes (PCPs) at single cell resolution. N-(2-Mercapto-propionyl)glycine-coated silver nanoparticles were synthesized by reduction of silver nitrate using sodium borohyride and then succinimidylated via ligand exchange. Alexa Fluor 647-labeled concanavalin A (con A) was chemically bound to the silver particles to make the fluorescent metal plasmon-coupled probes. The fluorescence images were collected using a scanning confocal microscopy. The fluorescence intensity was observed to enhance 7-fold when binding the labeled con A on a single silver particle. PCPs were conjugated on HEK 293 A cells. Imaging results demonstrate that cells labeled by PCPs were 20-fold brighter than those by free labeled con A.     

Determination of phosphatidylcholine monohydroperoxides using quadrupole time-of-flight mass spectrometry

An improved technique for the analysis of phosphatidylcholine (PC) monohydroperoxides was developed using quadrupole time-of-flight (Q-TOF) mass spectrometry with electrospray ionization. Separation was obtained using an HPLC C8 column with a gradient of methanol and 10 mM aqueous ammonium acetate. Monohydroperoxides of palmitoyl-linoleoyl (C16:0/C18:2) PC, stearoyl-linoleoyl (C18:0/C18:2) PC, and oleoyl-linoleoyl (C18:1/C18:2) PC were detected mainly as MH(+) and [M+Na](+) ions in the heart of the intact rat. Using standard synthetic PCOOH (C16:0/C18:2-OOH), the lipid extract component was identified as (C16:0/C18:2-OOH) PC based on the product ions of ESI-MS-MS and, the PCOOH concentration was quantitated using HPLC with chemiluminescence detection. Two epoxyhydroxy derivatives of the three PCs mentioned above were also detected. This is the first report to show the presence of monohydroperoxides and epoxyhydroxy-derivatives of (C16:0/C18:2)PC, (C18:0/C18:2)PC, and (C18:1/C18:2) PC in the rat heart.     

Determination of protease inhibitors using liquid chromatography-tandem mass spectrometry

A method for the analysis of six protease inhibitors and one metabolite has been developed and validated. Amprenavir, ritonavir, saquinavir, lopinavir, indinavir, nelfinavir, and an active metabolite of nelfinavir (M8) are quantitated using reversed-phase liquid chromatography coupled to tandem mass spectrometry, equipped with an electrospray ionization source (ESI-LC-MS-MS). The validation data presented here shows that the method allows the rugged analysis of these species from one aliquot. The evolution of complex drug interactions assessments and the clinical use of therapeutic drug monitoring for these antiretrovirals will be a potential immediate application of this method.     

Determination of benzimidazole residues using liquid chromatography and tandem mass spectrometry

The determination of residues of benzimidazole using liquid chromatography and tandem mass spectrometry (LC–MS–MS) with ion spray ionization is described. Swine muscle tissue was spiked with a mixture of fifteen benzimidazoles, including metabolites of fenbendazole and albendazole. As clean-up procedure, an ethyl acetate extraction followed by solid-phase extraction on styrol-divinyl-benzene cartridge was used. The evaluation was performed by selecting the characteristic product ions for the benzimidazoles and using multiple reaction mode. 2-n-Butylmercaptobenzimidazole was used as internal standard. Blank muscle samples were fortified in the concentration range of 1–22 μg/kg. The limits of detection were below 6 μg/kg and the limits of quantification for most benzimidazoles were below 10 μg/kg. The matrix effect was checked using spiked muscle tissues of cattle and sheep as well as liver of cattle. Practical application will be shown by incurred egg material from laying hens treated with flubendazole. The recovery of the clean-up was mostly above 50% in muscle tissue and 70% in egg yolk.     

Determination of small amounts of noradrenaline using a fluorescence histochemical method

Summary Using the Hayashi method, the distribution of glucosaminidase was studied throughout sequential molar development. Conspicuous activity was observed in certain cellular components of the stellate reticulum and dental papilla. The outer enamel epithelium, stratum intermedium and ameloblasts displayed less intense reactions.Supported by PHS Grant No. 2800-02 —Tooth Germ Development, National Institute of Dental Research, National Institutes of Health.Lieutenant Commander, Dental Corps, United States Navy.