Quantification of cells with specific phenotypes II: determination of CD4 expression level on reconstituted lyophilized human PBMC labelled with anti-CD4 FITC antibody.

This report focuses on the characterization of CD4 expression level in terms of equivalent number of reference fluorophores (ERF). Twelve different flow cytometer platforms across sixteen laboratories were utilized in this study. As a first step the participants were asked to calibrate the fluorescein isothiocyanate (FITC) channel of each flow cytometer using commercially available calibration standard consisting of five populations of microspheres. Each population had an assigned value of equivalent fluorescein fluorophores (EFF denotes a special case of the generic term ERF with FITC as the reference fluorophore). The EFF values were assigned at the National Institute of Standards and Technology (NIST). A surface-labelled lyophilized cell preparation was provided by the National Institute of Biological Standards and Control (NIBSC), using human peripheral blood mononuclear cells (PBMC) pre-labeled with a FITC conjugated anti-CD4 monoclonal antibody. Three PBMC sample vials, provided to each participant, were used for the CD4 expression analysis. The PBMC are purported to have a fixed number of surface CD4 receptors. On the basis of the microsphere calibration, the EFF value of the PBMC samples was measured to characterize the population average CD4 expression level of the PBMC preparations. Both the results of data analysis performed by each participant and the results of centralized analysis of all participants' raw data are reported. Centralized analysis gave a mean EFF value of 22,300 and an uncertainty of 750, corresponding to 3.3% (level of confidence 68%) of the mean EFF value. The next step will entail the measurement of the ERF values of the lyophilized PBMC stained with labels for other fluorescence channels. The ultimate goal is to show that lyophilized PBMC is a suitable biological reference cell material for multicolor flow cytometry and that it can be used to present multicolor flow cytometry measurements in terms of ABC (antibodies bound per cell) units.

Measurement of Microsphere Concentration Using a Flow Cytometer with Volumetric Sample Delivery.

Microsphere concentrations are needed to assign equivalent reference fluorophores (ERF) units to microspheres used in quantitative flow cytometry. A flow cytometer with a syringe based sample delivery system was evaluated for the measurement of the concentration of microspheres contained in a vial of lyophilized microspheres certified by BD Biosciences to contain 50,600 microspheres. The concentration was measured by counting the number of microspheres contained in the volume delivered by the flow cytometer and dividing the number by the volume. The syringe volume was calibrated both in the delivery and draw modes, and the results of the volume calibration were summarized by two calibration lines. The delivered volume was obtained by dividing the number of recorded events by the concentration of microsphere count standard in the sample tube. The draw volume was obtained by weighting the sample tube before and after the draw. The slope of the draw volume calibration line was equal to 1.00 with an offset of -13 µL. The slope of the delivered volume calibration was 0.93 suggesting a systematic volume-dependent bias, which can be rationalized as an effect of suspension flow in capillaries. When the sample volume was set to values between 150 µL and 300 µL, both calibration curves gave similar results suggesting that a good estimate of the true delivered volume can be obtained by subtracting 13 µL from the delivered volume indicated by the syringe settings. The number of microspheres in the volume was obtained by passing the suspension contained in the volume through a laser beam and counting the number of events in which the signals from the scattering and fluorescence detectors exceeded threshold values. Measurements were performed with the lyophilized microspheres made by BD Biosciences and fluorescein microspheres (expired reference material RM 8640) in three buffers: a phosphate buffer saline (PBS), a buffer containing PBS and 0.05 % BSA (bovine serum albumin) by mass, and a buffer containing PBS and 0.05 % TWEEN 20 detergent solution (P1379 Sigma-Aldrich) by mass. It was found that the concentration of count standard was significantly higher in the PBS+BSA buffer relative to the value obtained in PBS buffer. Values for PBS+0.05 % TWEEN 20 buffer were intermediate. The effect of buffer on the measured microsphere concentration was reported previously. The suggested procedure for the measurement of the concentration of microspheres with the flow cytometer is to use PBS+0.05 % BSA buffer, accumulate data for a delivered volume of 150 µL to 300 µL, and reduce the indicated delivered volume by 13 µL when performing the concentration calculation. The procedure was tested on a mixture of lyophilized microspheres and RM 8640 microspheres. The resulting lyophilized microsphere concentration was consistent with the certified value. The RM 8640 concentration determined using the suggested procedure was consistent with the concentration value determined using the relative method with the lyophilized microspheres as the reference. The uncertainties, obtained from one standard deviation of repeated measurements, were about 4 %.

Measurement of Scattering and Absorption Cross Sections of Microspheres for Wavelengths between 240 nm and 800 nm.

A commercial spectrometer with a 150 mm integrating sphere (IS) detector was used to estimate the scattering and absorption cross sections of monodisperse polystyrene microspheres suspended in water. Absorbance measurements were performed with the sample placed inside the IS detector. The styrene absorption was non zero for wavelengths less than 300 nm. Correction for fluorescence emission by styrene was carried out and the imaginary part of the index of refraction, ni, was obtained. Absorbance measurements with the sample placed outside the IS detector were sensitive to the loss of photons from the incident beam due to scattering. The absorbance data was fitted with Lorenz-Mie scattering cross section and a correction for the finite acceptance aperture of the spectrometer. The fit parameters were the diameter, the suspension concentration, and the real part of the index of refraction. The real part of the index was parameterized using an expansion in terms of powers of the inverse wavelength. The fits were excellent from 300 nm to 800 nm. By including the imaginary part obtained from the absorbance measurements below 300 nm, it was possible to obtain a good fit to the observed absorbance data over the region 240 nm to 800 nm. The value of ni at 266 nm was about 0.0060±0.0016 for microspheres with diameters of 1.5 µm, 2.0 µm, and 3.0 µm. The scattering cross section, absorption cross section, and the quantum yield at 266 nm of microsphere with a diameter of 2.0 µm was 5.65±0.01 µm(2), 1.54±0.03 µm(2), and 0.027±0.002 respectively. The styrene absorption reduces the scattering cross section by 20 % at 266 nm.

Measurement of Scattering Cross Section with a Spectrophotometer with an Integrating Sphere Detector.

A commercial spectrometer with an integrating sphere (IS) detector was used to measure the scattering cross section of microspheres. Analysis of the measurement process showed that two measurements of the absorbance, one with the cuvette placed in the normal spectrometer position, and the second with the cuvette placed inside the IS, provided enough information to separate the contributions from scattering and molecular absorption. Measurements were carried out with microspheres with different diameters. The data was fitted with a model consisting of the difference of two terms. The first term was the Lorenz-Mie (L-M) cross section which modeled the total absorbance due to scattering. The second term was the integral of the L-M differential cross section over the detector acceptance angle. The second term estimated the amount of forward scattered light that entered the detector. A wavelength dependent index of refraction was used in the model. The agreement between the model and the data was good between 300 nm and 800 nm. The fits provided values for the microsphere diameter, the concentration, and the wavelength dependent index of refraction. For wavelengths less than 300 nm, the scattering cross section had significant spectral structure which was inversely related to the molecular absorption. This work addresses the measurement and interpretation of the scattering cross section for wavelengths between 300 nm and 800 nm.

Measurement of Absorption and Scattering With an Integrating Sphere Detector: Application to Microalgae.

A spectrometer with an integrating sphere (IS) detector was used to measure the absorbance due to scattering and absorption. Analysis of the measurement process showed that two measurements of the absorbance, one with the cuvette placed in the normal spectrometer position, and the second with the cuvette placed next to the entrance aperture of the IS detector, provide enough information to separate the contributions from scattering and molecular absorption. Measurements were carried out with mixtures of microsphere and chromophore solutions. Two cases were examined: microspheres suspended in an aqueous fluorescein solution, and microspheres suspended in an aqueous holmium oxide solution. In both cases, the proposed measurement model gave results which were in good agreement with the expected response. Measurements on microalgae suspensions yielded a molecular absorption contribution and a scattering contribution. The scattering contribution had significant spectral structure which was inversely related to the molecular absorption contribution. The absorption and scattering contributions may provide independent information on the status of chlorophyll molecules and the structure of chloroplasts in microalgae.

Procedures for Wavelength Calibration and Spectral Response Correction of CCD Array Spectrometers.

This work describes a procedure for acquiring a spectrum of an analyte over an extended range of wavelengths and validating the wavelength and intensity assignments. To acquire a spectrum over an extended range of wavelengths with a spectrometer with a charge coupled device (CCD) array detector, it is necessary to acquire many partial spectra, each at a different angular position of the grating, and splice the partial spectra into a single extended spectrum. The splicing procedure exposes instrument dependent artifacts. It is demonstrated that by taking a spectrum of a reference irradiance source and making spectral correction, the artifacts exposed by the splicing are removed from the analyte spectrum. This is because the irradiance reference spectrum contains the same artifacts as the analyte spectrum. The artifacts exposed by the splicing depend on the wavelength of the splice; therefore it is important to measure the irradiance reference spectrum for the same range of wavelengths used to measure the spectrum of the analyte solution. In other words, there is no general spectral correction factor which is applicable to spectra taken for different range of wavelengths. The wavelength calibration is also carried out by splicing many partial spectra from a source like a krypton lamp. However the wavelength assignments are not sensitive to the splicing procedure and the same wavelength calibration can be used for spectra acquired over different extended wavelength ranges. The wavelength calibration checks the validity of the setting of the grating angular position, and the assignment of wavelengths to individual pixels on the CCD array detector. The procedure is illustrated by measuring the spectrum of an orange glass and the spectrum of a suspension of microalgae.

Evaluating the quality of data from DNA microarray measurements.

Gene expression technology offers great potentials to generate new insights into human disease pathogenesis; however, the data quality remains a major obstacle for realizing its potentials. In the present study 60-mers oligonucleotide target immobilized on coated glass slides were utilized as a model system to investigate parameters, such as target concentration, retention, signal linearity, and fluorescence properties of fluorophores, which likely affect the quality of microarray results. An array calibration slide was used to calibrate an Axon GenePix 4000A scanner and ensure the dynamic range of the instrument. The work is a first step toward our goal of quantitative gene expression measurements.

Monoclonal antibody selection for interleukin-4 quantification using suspension arrays and forward-phase protein microarrays.

A recombinant mouse interleukin-4 (IL-4) and three different purified rat antimouse IL-4 monoclonal antibodies (Mab) with different clonalities were employed as a model system. This system was used to examine monoclonal antibody effectiveness using both conventional and high-throughput measurement techniques to select antibodies for attaining the most sensitive detection of the recombinant IL-4 through the "sandwich-type" immunoassays. Surface plasmon resonance (SPR) measurements and two high-throughput methods, suspension arrays (also called multiplexed bead arrays) and forward-phase protein microarrays, predicted the same capture (BVD4-1D11) and detection (BVD6-24G2) antibody pair for the most sensitive detection of the recombinant cytokine. By using this antibody pair, we were able to detect as low as 2 pg/mL of IL-4 in buffer solution and 13.5 pg/mL of IL-4 spiked in 100% normal mouse serum with the multiplexed bead arrays. Due to the large amount of material required for SPR measurements, the study suggests that the multiplexed bead arrays and protein microarrays are both suited for the selection of numerous antibodies against the same analyte of interest to meet the need in the areas of systems biology and reproducible clinical diagnostics for better patient care.

Photophysical properties of ricin.

The molar absorption coefficient of ricin in phosphate-buffered saline (PBS) at 279 nm was measured as (93,900+/-3300) L mol(-1) cm(-1). The concentration of ricin was determined using amino acid analysis. The absorption spectrum of ricin was interpreted in terms of 69% contribution from absorption by tryptophan residues and 31% contribution from absorption by tyrosine residues. The total dipole strength of the ricin band at 280 nm was determined to be (147+/-8) D2 and was consistent with the combined dipole strengths of 10 tryptophan ([11.7+/-1.0] D2) and 23 tyrosine ([1.4+/-0.2] D2) residues. The structure of ricin was used to determine the coupling of the tryptophan residues in ricin. The maximum interaction energy was found to be 424 cm(-1)/epsilon while the average interaction between any two pairs of tryptophan residues was approximately 18 cm(-1)/epsilon. In this study, epsilon is the dielectric constant inside the protein. The fluorescence from ricin, excited at 280 nm, was dominated by fluorescence from tryptophan residues suggesting the presence of energy transfer from tyrosine to tryptophan residues. The absorbance and fluorescence of ricin increased slightly when ricin was denatured in a high concentration of guanidine. Irreversible thermal unfolding of ricin occurred between 65 degrees C and 70 degrees C. (D=3.3364*10(-30) Cm, not SI unit, convenient unit for the magnitude of the electric dipole moment of molecules.).

Biophotonic Tools in Cell and Tissue Diagnostics.

In order to maintain the rapid advance of biophotonics in the U.S. and enhance our competitiveness worldwide, key measurement tools must be in place. As part of a wide-reaching effort to improve the U.S. technology base, the National Institute of Standards and Technology sponsored a workshop titled "Biophotonic tools for cell and tissue diagnostics." The workshop focused on diagnostic techniques involving the interaction between biological systems and photons. Through invited presentations by industry representatives and panel discussion, near- and far-term measurement needs were evaluated. As a result of this workshop, this document has been prepared on the measurement tools needed for biophotonic cell and tissue diagnostics. This will become a part of the larger measurement road-mapping effort to be presented to the Nation as an assessment of the U.S. Measurement System. The information will be used to highlight measurement needs to the community and to facilitate solutions.

Modeling of Photochemical Reactions in a Focused Laser Beam.

Fluorescent materials play a prominent role in the qualitative and quantitative measurement of scientific phenomena of importance in biotechnology and biomedical applications. Photodegradation of fluorophores is a process that determines the accuracy and sensitivity of such measurements. This is the motivation for developing methods for accurately measuring fluorophore photodegradation rates. Recently, illumination consisting of short pulses has been used to examine the decay of photochemical reaction products. However, the time resolved measurements are difficult to interpret since the photodegradation process usually involves multiple time scales. The frequency domain measurement technique discussed here looks at the frequency response of a fluorescent sample to a frequency modulated illuminating light. The photodegradation rate is obtained by interpreting the frequency domain measurements in terms of traditional impedance concepts. In the measurements described in this paper, a focused laser beam is used to illuminate a sample of slowly flowing fluorescent solution. The laser beam is assumed to have a Gaussian power distribution hence illumination is spatially non-uniform in the region of interest. The photochemical reaction rates depend on power, so they will also vary with the position in the beam. However in the case of photodegradation of fluorophores, the measurement of the resulting decrease in fluorescence is given in terms of the radiation emitted from the entire illuminated region. In this work we present a mathematical description of the time evolution of the fluorescence response integrated over a non-uniformly illuminated domain. As a result of our analysis, an experimentally accessible and tractable mathematical model Eq. (19) and Eq. (30) is obtained from a more fundamental description given by Eq. (4) and Eq. (5). The model is used to create a functional form for fitting experimental measurements from a lock-in amplifier.

Comparison of ovalbumin quantification using forward-phase protein microarrays and suspension arrays.

We employed ovalbumin (a simulant used for ricin and botulism toxins in biodefense applications) and its high affinity polyclonal antibody as a model system to examine the sensitivity, dynamic range, linearity, and reproducibility of forward-phase array results in comparison to suspension arrays. It was found that protein microarrays had a dynamic range of 4 orders of magnitude and a sensitivity of less than 1 pg/mL, respectively. The dynamic range and sensitivity of suspension arrays were close to 2 orders of magnitude and 0.25 ng/mL, respectively. The sensitivity we observed for the suspension arrays is comparable to that reported for enzyme-linked immunosorbent assays (ELISAs) in the literature. We used ovalbumin samples with two different purities, 38.0% and 76.0% (w/w), as determined by polyacrylamide gel electrophoresis (PAGE). These samples were used to evaluate the effect of impure samples on detection. The data obtained from the forward-phase protein arrays gave values that were consistent with the PAGE data. The data from the suspension arrays were not as consistent and may indicate that this format may not give as reliable data with impure samples. Knowledge of the advantages and disadvantages of the two proteomic methods would allow their more rational use in clinical diagnosis.

Fluorescent nanometer microspheres as a reporter for sensitive detection of simulants of biological threats using multiplexed suspension arrays.

We succeeded in using 40 nm FRET (fluorescence resonance energy transfer) microspheres conjugated to antibodies as the fluorescent reporters to perform the multiplexing suspension array measurements on two simulants of biological threats, ricin (A chain) and a crude spore preparation of Bacillus globigii (Bg). The microspheres were impregnated with two types of fluorophores in equal number (approximately 140 fluorophores in total per microsphere) and displayed bright PE-like fluorescence via a fluorescence resonance energy transfer mechanism. Activated microspheres (aldehyde groups) were directly coupled to antibodies and used to form sandwich-type immunoassays in a suspension array. For the crude preparations of Bg, the assay sensitivity using antibody-conjugated microspheres is an order of magnitude higher than that using the conventional fluorescent reporter, R-phycoerythrin (PE). Using the microspheres, Bg at the concentration of 5 ng/mL can be easily detected. For ricin, the assay sensitivity was similar to that obtained using PE as the reporter, but washing the reaction mixtures resulted in the fluorescence signals that were 2-3 times higher compared to those using PE. Ricin at a concentration of 1 ng/mL can be readily identified. Importantly, the two simulants do not interfere with each other in the multiplexing experiments. The 40 nm FRET microspheres are a new sensitive alternative as fluorescent reporters for detection in suspension arrays.

Quantitating Fluorescence Intensity From Fluorophore: Assignment of MESF Values.

A procedure is presented to convert the comparison of measured fluorescence signals into a comparison of fluorescence yields (FY). The fluorescence yield, which is a property of a solution or a suspension, is defined as the product of the fluorophore concentration and the molecular quantum yield. The paper revises the measurement model which relates the measured fluorescence signal to the FY. The equality of FY of two solutions provides an equivalence between the concentrations of fluorophore in the two solutions. The equivalence is the basis for quantitation in terms of molecules of equivalent soluble fluorophore (MESF). The quantitation procedure starts with the measurement of fluorescence signals from a serial dilution of fluorescein solutions to obtain a calibration of a fluorometer. The fluorometer is used to measure the fluorescence signal of a suspension of microspheres with immobilized fluorescein isothiocyanate (FITC). The calibration is used to obtain the concentration of soluble fluorophores which gives the same fluorescence signal as the microsphere suspension. The number concentration of microspheres is measured and the equality of fluorescence yields is used to obtain the number of soluble fluorescein molecules equivalent to a single microsphere.

The effect of overhanging nucleotides on fluorescence properties of hybridising oligonucleotides labelled with Alexa-488 and FAM fluorophores.

In order to rationally select and design probes for real-time PCR, we have determined the influence of the overhang region of the complementary strand on the resulting fluorescence from a hybridising probe. A series of target oligonucleotides, each with a unique 3' overhang (4 bases), was hybridised to either 5' fluorescein (FAM)- or Alexa-488-labelled probes, and the changes in fluorescence properties were monitored. We found that the number of guanine bases in the overhang region of the target oligonucleotides was proportional to the amount of fluorescence quenching observed for both the FAM and Alexa-488 dyes. FAM appeared to be more sensitive to guanine-induced quenching with three and four guanine bases resulting in greater than a twofold decrease in the quantum yield of the fluorophore compared to the no-overhang target. In addition, we found that adenine bases caused fluorescence quenching of the Alexa-488-labelled probe, whereas the FAM-labelled probe appeared insensitive. The quenching data, generated with the steady-state fluorescence measurements, displayed a linear correlation with that obtained using a fluorescent thermal cycler, suggesting the applicability to real-time PCR measurements. Anisotropy data from the series of duplexes correlated with the fluorescence quantum yield, suggesting that quenching was accompanied by increased dye mobility.

Spectroscopic characterization of fluorescein- and tetramethylrhodamine-labeled oligonucleotides and their complexes with a DNA template.

We measured absorption and emission spectra, fluorescence quantum yield, anisotropy, fluorescence resonance energy transfer (FRET), and melting temperature to characterize fluorescein- and tetramethylrhodamine (TMR)-labeled oligonucleotides in solution and when hybridized to a common DNA template. Upon hybridization to the template, both the absorption and emission spectra of TMR-labeled duplexes exhibited a shift with respect to those of labeled oligonucleotides, depending on the location of the TMR on the oligonucleotide. Measurements of quantum yield, anisotropy, and melting temperature indicated that TMR interacted with nucleotides within the duplexes in the order (T1>T5>T11, T16) that the oligonucleotide with TMR labeled at the 5' end (T1) is stronger than that labeled at position 5 from the 5' end (T5), which is also stronger than those labeled at the positions, 11 and 16, from the 5' end (T11, T16). In the case of the duplex formed between T1 and the template, fluorescence quenching was observed, which is attributed to the interaction between the dye molecule and guanosines located at the single-stranded portion of the template. A two-state model was suggested to describe the conformational states of TMR in the duplex. The melting temperatures of the four FRET complexes show the same pattern as those of TMR-labeled duplexes. We infer that the interactions between TMR and guanosine persist in the FRET complexes. This interaction may bring the donor and the acceptor molecules closely together, which could cause interaction between the two dye molecules shown in absorbance measurements of the FRET complexes.

Fluorescence resonance energy transfer between donor-acceptor pair on two oligonucleotides hybridized adjacently to DNA template.

We use fluorescein as the energy donor and rhodamine as the acceptor to measure the efficiency of fluorescence resonance energy transfer (FRET) in a set of hybridized DNA constructs. The two fluorophores are covalently attached via linkers to two separate oligonucleotides with fluorescein at the 3' end of one oligonucleotide and rhodamine at the 5' end or in the middle of another nucleotide. For the FRET analysis both fluorophore-labeled oligonucleotides are hybridized to adjacent sections of the same DNA template to form a three-component duplex with a one base gap between the two labeled oligonucleotides. A similar configuration is implemented for a quantitative real-time polymerase chain reaction (PCR) with LightCycler technology, where a 1-5 base separation between donor and acceptor is recommended to optimize energy transfer efficiencies. Our constructs cover donor-acceptor separations from 2 to 17 base pairs (approximately 10-70 A). The results show that, when the two fluorophores are located at close distances (less than 8 base separation), FRET efficiencies are above 80%, although there may be ground-state interactions between fluorophores when the separation is under about 6 bases. Modeling calculations are used to predict the structure of these three-component constructs. The duplex mostly retains a normal double helical structure, although slight bending may occur near the unpaired base in the DNA template. Stable and reproducible energy transfer is also observed over the distance range investigated here in real-time thermal cycling. The study identifies important parameters that determine FRET response in applications such as real-time PCR.

Frequency-domain measurement of the photodegradation process of fluorescein.

The frequency-domain technique is applied to measure the photodegradation rate of fluorescein in aqueous solutions. The illuminating light is modulated, and the changes in fluorescence from the illuminated region are detected synchronously. A constant flow rate is imposed on the fluorescein solution to control the mass transport of fluorescein into the illuminated region. The fluorescence response is described by a model that assumes that photodegradation occurs from the triplet excited state. The predictions of the model are consistent with the observed variations in the fluorescence response with flow rate, modulation frequency and incident power. We discuss in this article how the dependence of the model parameters on experimental conditions can be used to infer the photodegradation rate as well as some of the details of the photodegradation mechanism. The results are consistent with the known mechanism of photodegradation of fluorescein. The frequency-domain technique gives a photodegradation rate of 53 s(-1) in an air-saturated solution and 37 s(-1) in solutions purged with argon gas.

Application of electromodulated fluorescence to the study of the dynamics of single-strand oligonucleotides at modified gold surfaces.

Single-strand oligonucleotides with fluorescein labels were immobilized on modified gold electrodes and the surface fluorescence intensity was measured as a function of the potential applied to the electrode. The potential consisted of a constant and sinusoidal parts. The fluorescence was detected synchronously with the sinusoidal component of the potential. Large variation in the electromodulated fluorescence (EmF) was observed as a function of the constant component of the applied potential. In the potential range -0.2 to +0.2 V (vs Ag/AgCl) the EmF signal was quasi-reversible. A model for surface fluorescence was applied to the analysis of the EmF in this potential range. The model assumed that the adsorption-desorption process and quenching by the metal were the dominant mechanisms responsible for the observed EmF. For constant potential more negative than -0.5 V, the observed EmF response appeared to have a contribution from ssDNA in solution. This interpretation was suggested by comparison to EmF from solutions containing free fluorescein or labeled ssDNA. Large variation in the EmF response was observed for electrodes modified with amine and carboxyl groups. This suggests that electrostatic interactions play a dominant role.

Raman and FTIR spectroscopies of fluorescein in solutions.

Raman and Fourier transform-infra red (FT-IR) spectroscopies of fluorescein in aqueous solutions have been investigated in the pH range from 9.1 to 5.4. At pH 9.1 fluorescein is in the dianion form. At pH 5.4, fluorescein is a mixture of monoanion (approximately 85%), dianion and neutral forms (together approximately 15%). The fluorescence quantum yield drops from 0.93 for the dianion form to 0.37 for the monoanion form. The Raman and FT-IR studies focused on the frequency range from 1000 to 1800 cm(-1) which contains the skeletal vibrational modes of the xanthene moiety of fluorescein. At pH 9.1, the spectroscopic feature of fluorescein dianion are consistent with a picture of an electron delocalized among the xanthene moiety and two identical oxygens attached to opposite ends of the xanthene moiety, forming a very symmetric structure. The characteristic of fluorescein dianion is the presence of the phenoxide-like stretch at 1310 cm(-1). At pH 5.4, fluorescein monoanion has lost the symmetric structure characteristic of the dianion. The spectra of the monoanion have distinctive contributions from the phenolic bend at 1184 cm(-1). The assignments of the vibrational bands shown in Raman and FT-IR spectra are given based on both literature and the ab initio calculations at the Hartree-Fock level with HF/6-31 + +G* basis set. Excellent correlation is found between the experimental and calculated spectra.