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Pigments and melanins of fungal spores have been investigated for decades, revealing important roles in the survival of the fungus in hostile environments. The key genes and the encoded enzymes for pigment and melanin biosynthesis have recently been found in Ascomycota, including Aspergillus spp. In Aspergillus terreus , the pigmentation has remained mysterious with only one class of melanin biogenesis being found. In this study, we examined an intriguing, partially annotated gene cluster of A. terreus strain NIH2624, utilizing previously sequenced transcriptome and improved gene expression data of strain MUCL 38669, under the influence of a suggested quorum sensing inducing metabolite, butyrolactone I. The core polyketide synthase (PKS) gene of the cluster was predicted to be significantly longer on the basis of the obtained transcriptional data, and the surrounding cluster was positively regulated by butyrolactone I at the late growth phase of submerged culture, presumably during sporulation. Phylogenetic analysis of the extended PKS revealed remarkable similarity with a group of known pigments of Fusarium spp., indicating a similar function for this PKS. We present a hypothesis of this PKS cluster to biosynthesise a 1,8-dihydroxynaphthalene (DHN)-type of pigment during sporulation with the influence of butyrolactone I under submerged culture.

Filamentous fungi of the Ascomycota phylum are known to contain a family of conserved conidiation regulating proteins with distinctive velvet domains. In Aspergilli, this velvet family includes four proteins, VeA, VelB, VelC and VosA, and is involved in conidiation and secondary metabolism along with a global regulator LaeA. In A. terreus, the overexpression of LaeA has been observed to increase the biogenesis of the pharmaceutically-important secondary metabolite, lovastatin, while the role of the velvet family has not been studied. The secondary metabolism and conidiation of A. terreus have also been observed to be increased by butyrolactone I in a quorum-sensing manner. An enlightenment of the interplay of these regulators will give potential advancement to the industrial use of this fungus, as well as in resolving the pathogenic features. In this study, the Aspergillus terreus MUCL 38669 transcriptome was strand-specifically sequenced to enable an in-depth gene expression analysis to further investigate the transcriptional role of butyrolactone I in these processes. The sequenced transcriptome revealed intriguing properties of the velvet family transcripts, including the regulator laeA, and uncovered the velC gene in A. terreus. The reliability refining microarray gene expression analysis disclosed a positive regulatory role for butyrolactone I in laeA expression, as well as an influence on the expression of the canonical conidiation-regulating genes under submerged culture. All of this supports the suggested regulative role of butyrolactone I in A. terreus secondary metabolism, as well as conidiation.

Background: Atherosclerosis is a complex disease with hundreds of genes influencing its progression. In addition, the phenotype of the disease varies significantly depending on the arterial bed., Methodology/principal Findings: We characterized the genes generally involved in human advanced atherosclerotic (AHA type V-VI) plaques in carotid and femoral arteries as well as aortas from 24 subjects of Tampere Vascular study and compared the results to non-atherosclerotic internal thoracic arteries (n=6) using genome-wide expression array and QRT-PCR. In addition we determined genes that were typical for each arterial plaque studied. To gain a comprehensive insight into the pathologic processes in the plaques we also analyzed pathways and gene sets dysregulated in this disease using gene set enrichment analysis (GSEA). According to the selection criteria used (>3.0 fold change and p-value <0.05), 235 genes were up-regulated and 68 genes down-regulated in the carotid plaques, 242 genes up-regulated and 116 down-regulated in the femoral plaques and 256 genes up-regulated and 49 genes down-regulated in the aortic plaques. Nine genes were found to be specifically induced predominantly in aortic plaques, e.g., lactoferrin, and three genes in femoral plaques, e.g., chondroadherin, whereas no gene was found to be specific for carotid plaques. In pathway analysis, a total of 28 pathways or gene sets were found to be significantly dysregulated in atherosclerotic plaques (false discovery rate [FDR] <0.25)., Conclusions: This study describes comprehensively the gene expression changes that generally prevail in human atherosclerotic plaques. In addition, site specific genes induced only in femoral or aortic plaques were found, reflecting that atherosclerotic process has unique features in different vascular beds.

Besides the typical short-lived fluorescence with decay times in the nanosecond range, colloidal II/VI semiconductor nanoparticles dispersed in buffer also possess a long-lived fluorescence component with decay times in the microsecond range. Here, the signal intensity of the long-lived luminescence at microsecond range is shown to increase 1,000-fold for CdTe nanoparticles in PBS buffer. This long-lived fluorescence can be conveniently employed for time-gated fluorescence detection, which allows for improved signal-to-noise ratio and thus the use of low concentrations of nanoparticles. The detection principle is demonstrated with a time-resolved fluorescence immunoassay for the detection of C-reactive protein (CRP) using CdSe-ZnS nanoparticles and green light excitation.

Background and Aims: Carbonic anhydrases (CA) play a central role in osteoclast function and bone remodeling by catalyzing the formation of bicarbonate and proton from carbon dioxide. According to previous histochemical studies, advanced atherosclerotic plaques share similarities with bone. However, whether CAs are expressed in plaques is not known., Methods and Results: Whole genome expression array of arterial samples (n = 24) confirmed that several genes indicating osteoblastogenesis and osteoclastogenesis were up-regulated in plaques when compared to control vessel samples from internal thoracic arteries (n = 6), including CA2 and CA12, expression of which was also verified with quantitative reverse transcription polymerase chain reaction (RT-PCR). In atherosclerotic plaques there was 11.6-fold (P < 0.0001) and 11.4-fold (P < 0.0001) up-regulation of CA2 and CA12, compared to controls, respectively. According to quantitative PCR, CA2 expression was elevated in carotid (12.3-fold, P < 0.0001), femoral (13.2-fold, P < 0.01), and aortic plaques (7.5-fold, P < 0.0001). CA12 expression was elevated in carotid (11.6-fold, P < 0.0001), femoral (11.5-fold, P < 0.01), and aortic plaques (9.7-fold, P < 0.0001). CAII, CAXII, and CD68 and tartrate-resistant acid phosphatase (TRAP), a marker of osteoclast-like cells, were found to be co-localized in multinucleated giant cells in the atherosclerotic plaques using immunohistochemistry and double-staining immunofluorescence analysis., Conclusions: The present findings provide evidence for the involvement of CAs in advanced atherosclerosis in osteoclast-like cells of monocyte-macrophage lineage.

Objective: We aimed to characterize the expression of indoleamine 2,3-dioxygenase (IDO) or IDO-induced tryptophan degradation-dependent pathways, which may lead to suppression of T cells and possible protection against atherosclerosis., Methods and Results: Expression of IDO and IDO-related pathway components was analyzed in advanced human atherosclerotic plaques (n = 24) and in non-atherosclerotic arteries (n = 6). Up-regulation of IDO and genes related to the IDO pathway was found to be pronounced in atherosclerotic plaques. Immunohistochemistry demonstrated IDO protein in the atheromatous core and co-distribution with monocyte-macrophages (CD68-positive cells). In gene-set enrichment analysis, the IDO pathway revealed a significant (false discovery rate (FDR) = 0.07) regulatory T cell, fork-head box protein 3 (FoxP3)-initiated CD28-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4)-inducible T cell co-stimulator (ICOS)-driven pathway leading to activation of IDO expression in antigen-presenting cells (APCs). Expression of these IDO pathway genes varied between 2.1- and 16.8-fold as compared to control tissues (P < 0.05 for all)., Conclusions: IDO and the IDO-related pathway are important mediators of the immunoinflammatory responses in advanced atherosclerosis offering new viable therapeutic targets for the development of antiatherogenic immunosuppressive therapies.

Introduction: Tumor grade is one of the most important prognostic factors in endometrioid endometrial adenocarcinoma. Amplification of oncogenes, such as Her2/neu, or loss of function of tumor suppressor genes, such as p53, are known to be associated with poor prognosis, but additional factors influencing clinical behavior are likely to exist. To examine the biological differences between low-grade and high-grade endometrioid endometrial adenocarcinomas, we compared gene expression in these 2 types of tumors., Methods: Six well-differentiated adenocarcinomas and 7 poorly differentiated adenocarcinomas were studied with 2 different microarray platforms, Affymetrix and Illumina. The expression of the most differentially expressed gene on both platforms was further studied in 34 endometrial adenocarcinoma samples (10 well differentiated, 9 moderately differentiated, and 15 poorly differentiated) using real-time reverse transcription-polymerase chain reaction., Results: The most differentially expressed gene on both platforms was Apolipoprotein E (APOE). In the poorly differentiated adenocarcinomas, APOE was overexpressed 13.1-fold (P = 0.001) and 9.7-fold (P = 0.007) when compared with well- and moderately differentiated tumors, respectively. There was no difference in APOE expression between well- and moderately differentiated adenocarcinomas., Conclusions: Increased expression of APOE might represent a late event in the progression of well-differentiated endometrioid endometrial adenocarcinoma to a poorly differentiated endometrioid endometrial adenocarcinoma. Although increased APOE expression has been previously reported in other malignancies, this is the first study to suggest that APOE might also have a role in endometrioid endometrial cancer.

Background and Aims: The expression of disintegrin and metalloprotease ADAM-9, ADAM-15, and ADAM-17 has been associated with cell-cell, cell-platelet, and cell-matrix interactions and inflammation. They are possibly implicated in the pathophysiology of atherosclerosis., Methods and Results: Whole-genome expression array and quantitative real-time polymerase chain reaction (PCR) analysis confirmed that ADAM-9, ADAM-15, and ADAM-17 are upregulated in advanced human atherosclerotic lesions in samples from carotid, aortic, and femoral territories compared to samples from internal thoracic artery (ITA) free of atherosclerotic plaques. Western analysis indicated that the majority of these ADAMs were in the catalytically active form. ADAM-9, ADAM-15, and ADAM-17-expressing cells were shown to co-localize with CD68-positive cells of monocytic origin in the atherosclerotic plaques using immunohistochemistry and double-staining immunofluorescence analysis. Co-localization was demonstrated in all vascular territories. In the carotid territory, cells expressing the ADAMs co-distributed also with smooth muscle cells and, in femoral territory, with CD31-positive endothelial cells, indicating that the ADAM expression pattern depends on vascular bed territory., Conclusions: Present findings provide strong evidence for the involvement of catalytically active ADAM-9, ADAM-15, and ADAM-17 in advanced atherosclerosis, most notably associated with cells of monocytic origin.

Objective: Previously, we scanned all 23,000 human genes for differential expression between normal and atherosclerotic tissues and found the involvement of ADAM8., Methods: We investigated the expression of ADAM8 mRNA and protein level in human atherosclerotic tissues and non-atherosclerotic internal thoracic arteries as well as the association of ADAM8 2662 T/G single nucleotide polymorphism (SNP) with the extent of coronary atherosclerosis and with the risk of fatal myocardial infarction., Results: ADAM8 mRNA was up-regulated in carotid, aortic, and femoral atherosclerotic plaques (n=24) when compared with non-atherosclerotic arteries. ADAM8 protein expression was increased in advanced atherosclerotic plaques as compared to control vessels wherein it was localized to macrophages and smooth muscle cells The G allele carriers of the ADAM8 2662 T/G SNP had significantly larger areas of fibrotic, calcified, and complicated plaques in coronary arteries (P=0.027, P=0.011, and P=0.011, respectively) and significantly higher occurrence of myocardial infarction (MI) (P=0.004) and fatal pre-hospital MI (P=0.003) than did the TT homozygotes., Conclusion: ADAM8 is a promising candidate to be involved in atherosclerosis, and its 2662 T/G allelic variant significantly associates with advanced atherosclerotic lesion areas and MI.

Background: Disturbed cellular cholesterol homeostasis may lead to accumulation of cholesterol in human atheroma plaques. Cellular cholesterol homeostasis is controlled by the sterol regulatory element-binding transcription factor 2 (SREBF-2) and the SREBF cleavage-activating protein (SCAP). We investigated whole genome expression in a series of human atherosclerotic samples from different vascular territories and studied whether the non-synonymous coding variants in the interacting domains of two genes, SREBF-2 1784G>C (rs2228314) and SCAP 2386A>G, are related to the progression of coronary atherosclerosis and the risk of pre-hospital sudden cardiac death (SCD)., Methods: Whole genome expression profiling was completed in twenty vascular samples from carotid, aortic and femoral atherosclerotic plaques and six control samples from internal mammary arteries. Three hundred sudden pre-hospital deaths of middle-aged (33-69 years) Caucasian Finnish men were subjected to detailed autopsy in the Helsinki Sudden Death Study. Coronary narrowing and areas of coronary wall covered with fatty streaks or fibrotic, calcified or complicated lesions were measured and related to the SREBF-2 and SCAP genotypes., Results: Whole genome expression profiling showed a significant (p = 0.02) down-regulation of SREBF-2 in atherosclerotic carotid plaques (types IV-V), but not in the aorta or femoral arteries (p = NS for both), as compared with the histologically confirmed non-atherosclerotic tissues. In logistic regression analysis, a significant interaction between the SREBF-2 1784G>C and the SCAP 2386A>G genotype was observed on the risk of SCD (p = 0.046). Men with the SREBF-2 C allele and the SCAP G allele had a significantly increased risk of SCD (OR 2.68, 95% CI 1.07-6.71), compared to SCAP AA homologous subjects carrying the SREBF-2 C allele. Furthermore, similar trends for having complicated lesions and for the occurrence of thrombosis were found, although the results were not statistically significant., Conclusion: The results suggest that the allelic variants (SREBF-2 1784G>C and SCAP 2386A>G) in the cholesterol homeostasis regulating SREBF-SCAP pathway may contribute to SCD in early middle-aged men.

Application of two-photon excitation of fluorescence in microscopy is one of the major discoveries of the "renaissance" of light microscopy that started in the 1980s. The technique derives its advantages from the biologically "smooth" wavelength of the excitation light and the confinement of the excitation. Difficult, and seemingly nontransparent, samples may be imaged with the technique with good resolution. Although the bioresearch has been concentrating mostly on the positive properties of the technique for imaging, the same properties may be applied successfully to nonimaging bioassays. This article focuses on the development path of two-photon excitation-based assay system.

Background: Bordetella pertussis is a gram-negative bacterium that infects the human respiratory tract and causes pertussis or whooping cough. The disease has resurged in many countries including Finland where the whole-cell pertussis vaccine has been used for more than 50 years. Antigenic divergence has been observed between vaccine strains and clinical isolates in Finland. To better understand genome evolution in B. pertussis circulating in the immunized population, we developed an oligonucleotide-based microarray for comparative genomic analysis of Finnish strains isolated during the period of 50 years., Methodology/principal Findings: The microarray consisted of 3,582 oligonucleotides (70-mer) and covered 94% of 3,816 ORFs of Tohama I, the strain of which the genome has been sequenced. Twenty isolates from 1953 to 2004 were studied together with two Finnish vaccine strains and two international reference strains. The isolates were selected according to their characteristics, e.g. the year and place of isolation and pulsed-field gel electrophoresis profiles. Genomic DNA of the tested strains, along with reference DNA of Tohama I strain, was labelled and hybridized. The absence of genes as established with microarrays, was confirmed by PCR. Compared with the Tohama I strain, Finnish isolates lost 7 (8.6 kb) to 49 (55.3 kb) genes, clustered in one to four distinct loci. The number of lost genes increased with time, and one third of lost genes had functions related to inorganic ion transport and metabolism, or energy production and conversion. All four loci of lost genes were flanked by the insertion sequence element IS481., Conclusion/significance: Our results showed that the progressive gene loss occurred in Finnish B. pertussis strains isolated during a period of 50 years and confirmed that B. pertussis is dynamic and is continuously evolving, suggesting that the bacterium may use gene loss as one strategy to adapt to highly immunized populations.

A method is introduced for quantitative detection of cell surface protein expression. The method is based on immunocytochemistry, the use of long decay time europium(III) chelate and platinum(II) porphyrin labels, and detection of photoluminescence emission from adhered cells by time-resolved fluorimetry. After immunocytochemistry, the assay wells are evaporated to dryness and measured in the dry state. This protocol allows repeated and postponed analysis and microscopy imaging. In order to investigate the performance of the method, we chose expression of intercellular adhesion molecule-1 (ICAM-1) of endothelial cell line EAhy926 as a research target. The expression of ICAM-1 on the cells was enhanced by introduction of a cytokine, tumour necrosis factor-alpha (TNFalpha). The method gave signal:background ratios (S:B) of 20 and 9 for europium and platinum labels, respectively, whereas prompt fluorescent FITC label gave a S:B of 3. Screening window coefficients (=Z'-factor) were >0.5 for all the three labels, thus indicating a score for an excellent screening assay. In conclusion, the method appears to be an appropriate choice for protein expression analysis, both in high-throughput screening applications, and for detailed sample investigation by fluorescent microscopy imaging., (Copyright (c) 2007 John Wiley & Sons, Ltd.)

In this paper, the challenging problem of robust mean-signal estimation of a single-step microparticle bioaffinity assay is investigated. For this purpose, a density estimation-based robust algorithm (DER) was developed. The DER algorithm was comparatively evaluated with four other parameter estimation methods (mean value, median filtering, least square estimation, Welsch robust m-estimator). Two important questions were raised and investigated: 1) Which of the five methods can robustly estimate the mean bioaffinity signal? and 2) How many microparticles need to be measured in order to obtain an accurate estimate of the mean signal value? To answer the questions, bootstrap and coefficient of variation (CV) analyses were performed. In the CV analysis, the DER algorithm gave the best results: The CV ranged from 0.8% to 4.9% when the number of microparticles used for the mean signal estimation varied from 800 to 30. In the bootstrap analysis of the standard error, the DER algorithm had the smallest variance. As a conclusion, it can be underlined that: 1) of all methods tested, the DER algorithm gave the most consistent and reproducible results according to the bootstrap and CV analysis; 2) using the DER algorithm accurate estimates could be calculated based on 80-100 particles, corresponding to a typical assay measurement time of 1 min; and 3) the investigated bioaffinity signals contained a large number of outliers (observations that severely deviate from the majority of data) and therefore robust techniques were necessary for the mean signal estimation tasks.

The reliability of rapid immunoassay is a concern due to an incomplete incubation to a non-equilibrium state and is susceptible to different error factors causing variance. The most critical point in the process should be found in order to improve the accuracy, and reproducibility of immunoassays, and enhance the system robustness. In this paper, the behavior of rapid assays is predicted by simulations using mechanistic assay model, based on antibody-analyte binding reaction kinetics. This antibody-analyte binding reaction kinetics model was constructed for a generic three-component (immunometric) assay and the parameters were chosen to be those of a known surface binding assay. The effects of the exact incubation timing and the initial reagent concentrations were studied focusing on the early phase of incubation, the non-equilibrium state. The magnitudes of errors in the input parameters were estimated using knowledge from practical immunoassays. According to simulations, inaccurate incubation timing adds error in the results at very short incubation times, especially in low analyte concentrations. The inaccurate reagent concentrations increase variance at short incubation times, as well. The error decreases rapidly after the first few minutes of incubation.

Fluorescence polarization is one of the most commonly used homogeneous assay principles in drug discovery for screening of potential lead compounds. In this article, the fluorescence polarization technique is combined with 2-photon excitation of fluorescence. Theoretically, the use of 2-photon excitation of fluorescence increases the volumetric sensitivity and polarization contrast of fluorescence polarization assays. The work in this report demonstrates these predictions for an estrogen receptor ligand binding assay.

A new separation-free method for detection of single nucleotide polymorphisms (SNPs) is described. The method is based on the single base extension principle, fluorescently labeled dideoxy nucleotides and two-photon fluorescence excitation technology, known as ArcDia trade mark TPX technology. In this assay technique, template-directed single base extension is carried out for primers which have been immobilized on polymer microparticles. Depending on the sequence of the template DNA, the primers are extended either with a labeled or with a non-labeled nucleotide. The genotype of the sample is determined on the basis of two-photon excited fluorescence of individual microparticles. The effect of various assay condition parameters on the performance of the assay method is studied. The performance of the new assay method is demonstrated by genotyping the SNPs of human individuals using double-stranded PCR amplicons as samples. The results show that the new SNP assay method provides sensitivity and reliability comparable to the state-of-the-art SNaPshot trade mark assay method. Applicability of the new method in routine laboratory use is discussed with respect to alternative assay techniques.

We describe the use of fluorophore-doped nanoparticles as reporters in a recently developed ArcDia TPX bioaffinity assay technique. The ArcDia TPX technique is based on the use of polymer microspheres as solid-phase reaction carrier, fluorescent bioaffinity reagents, and detection of two-photon excited fluorescence. This new assay technique enables multiplexed, separation-free bioaffinity assays from microvolumes with high sensitivity. As a model analyte we chose C-reactive protein (CRP). The assay of CRP was optimized for assessment of CRP baseline levels using a nanoparticulate fluorescent reporter, 75 nm in diameter, and the assay performance was compared to that of CRP assay based on a molecular reporter of the same fluorophore core. The results show that using fluorescent nanoparticles as the reporter provides two orders of magnitude better sensitivity (87 fM) than using the molecular label, while no difference between precision profiles of the different assay types was found. The new assay method was applied for assessment of baseline levels of CRP in sera of apparently healthy individuals.

Microspheres provide a solid phase substrate for bioaffinity binding similar to the walls of traditional test tubes and the wells of microtiter plates. The coated microsphere concentrates analyte molecules in the reaction volume on its surface. When the bioaffinity binding reaction has reached an equilibrium, the local concentration of the analyte in close proximity of the microsphere is orders of magnitude higher than the concentration of the analyte in the total reaction volume. The preparation and quality control of microspheres coated with bioactive material is less costly and labour intensive when compared to test tube or microwell plate coating procedures. In addition, the cost for logistics and transportation of microsphere reagents is lower than that of coated tubes or plates. Moreover, microspheres can be easily used in miniaturised assay formats and several different detection schemes can be employed in the measurement of microsphere-based assays. Several different types of microspheres are commercially available. The microspheres can be manufactured in different sizes from many materials, such as polystyrene, acrylate, and glass. The surface of the microspheres can be activated to enable covalent binding of biomolecules. Further, the microspheres may contain internal fluorochrome or magnetic material, for identification or separation purposes. In this paper we review different assay formats for single-step measurement of bioaffinity assays employing microspheres. The term single-step is used to describe assays where all reagents and the sample are mixed, incubated and measured without separate washing steps.

Phosphorescent conjugates of antibodies, neutravidin, and biotin (pentylamine derivative) were synthesized using previously described monofunctional labeling reagent of platinum(II) coproporphyrin-I with isothiocyanate reactive group (PtCP-NCS). These conjugates, which can be considered as standard reagents for a range of bioanalytical applications, were evaluated in solid-phase immunoassay schemes with the clinical analyte a-fetoprotein (AFP). A custom-designed time-resolved phosphorescence plate reader based on a compact and low-cost 532-nm laser and optimized for measurement of porphyrin labels was used. Using optimized tracers, instrumentation and assay protocols, subpicomolar detection limits were obtained both for PtCP label in solution and for AFP in solid-phase immunoassay. This sensitivity is comparable with standard time-resolved fluorescence immunoassays with lanthanide labels. The performance of metalloporphyrin labels, instrumentation, and solid-phase immunoassays as an alternative to the established detection platforms is discussed.

Recent developments in infrared laser technology have enabled the design of a compact instrumentation for two-photon excitation microparticle fluorometry (TPX). The microparticles can be used in immunoassays as the antibody-coated solid phase to capture an antigen and then detect it with a fluorescently labeled tracer antibody. Unlike most other methods, TPX technology allows low-volume, homogeneous immunoassays with real-time measurements of assay particles in the presence of a moderate excess of fluorescent tracer. In this study, the TPX assay system was used for the reagent characterization and the measurement of C-reactive protein (CRP) in diluted plasma samples, targeting the assay range useful in infectious disease diagnosis. The pentameric structure of the CRP permitted the optimization of an assay with the lowest detectable concentration of 1 microg/L (7.5 pM) by using a single monoclonal antibody both for capture and as the tracer. With a 1:200 predilution of samples, the measurement range of the assay was 1-150 mg/L, but an additional 1:10 dilution was required for higher concentrations. The TPX method showed a good correlation with the reference result obtained in a routine hospital laboratory, demonstrating the feasibility of the technology for immunodiagnostic applications.

At present, hemoglobin concentration and the volume of an erythrocyte can be determined from the intensities of light scattered by an individual cell at fixed angular intervals. This method is used in modern hemoglobin analyzers, but it requires calibration of optical and electronic units by certified particles of known size and refractive index. We describe a method that is based on the parametric solution of an inverse light-scattering problem and does not require a calibration procedure. The method is based on the use of parameters of the entire angular light-scattering pattern, called an indicatrix here. These parameters do not depend on the absolute intensity of light scattering. The indicatrix parameters form approximating equations that relate these parameters to the size and the phase-shift parameters of the particle. The applicability of the method is demonstrated by measurement of the indicatrices of individual sphered erythrocytes. The indicatrices of the individual erythrocytes were measured with a scanning flow cytometer at an angular range of from 15 to 55 deg. The volume and the hemoglobin concentration have been calculated by use of the developed method and by fitting of the experimental indicatrices to the indicatrices calculated from the Mie theory.

A statistical approach is presented to model the kinetics of cell distribution in the process of ligand-receptor binding on cell surfaces. The approach takes into account the variation of the amount of receptors on cells assuming the homogeneity of monovalent binding sites and ligand molecules. The analytical expressions for the kinetics of cell distribution have been derived in the reaction-limited approximation. In order to demonstrate the applicability of the mathematical model, the kinetics of binding the rabbit, anti-mouse IgG with Ig-receptors of the murine hybridoma cells has been measured. Anti-mouse IgG was labeled with fluorescein isothiocyanate (FITC). The kinetics of cell distribution on ligand-receptor complexes was observed during the reaction process by real-time measuring of the fluorescence and light-scattering traces of individual cells with the scanning flow cytometer. The experimental data were fitted by the mathematical model in order to obtain the binding rate constant and the initial cell distribution on the amount of receptors., (Copyright 2000 Academic Press.)

Background: Flow cytometry is a powerful tool for the analysis of individual particles in a flow. Differential light scattering (an indicatrix) was used for many years to obtain morphologic information about microorganisms. The indicatrices play the same role for individual particle recognition as a spectrum for substance characterization. We combined two techniques to analyze the indicatrix of the cells for the purpose of developing a database of light-scattering functions of cells., Methods: The scanning flow cytometer (SFC) allows the measurement of the entire indicatrix of individual particles at polar angles ranging from 5 degrees to 100 degrees. In this work, light-scattering properties of Escherichia coli have been studied both experimentally and theoretically with the SFC and the T-matrix method, respectively. The T-matrix method was used because of the nonspherical shape of E. coli cells, which were modeled by a prolate spheroid., Results: The indicatrices of E. coli cells were stimulated with T-matrix method at polar angles ranging from 10 degrees to 60 degrees. The absolute cross-section of light scattering of E. coli has been determined comparing the cross section of polystyrene particles modeled by a homogeneous sphere. The E. coli indicatrices were compared for logarithmic and stationary phases of cell growth., Conclusions: The indicatrices of E. coli were reproducible and could be used for identification of these cells in biologic suspensions. The angular location of the indicatrix minimum can be used in separation of cells in logarithmic and stationary phases. To use effectively the indicatrices for that purpose, the light-scattering properties of other microorganisms have to be studied., (Copyright 2000 Wiley-Liss, Inc.)

Two-photon fluorescence excitation has been found to be a very powerful method for enhancing the sensitivity and resolution in far-field light microscopy. Two-photon fluorescence excitation also provides a substantially background-free detection on the single-molecule level. It allows direct monitoring of formation of labelled biomolecule complexes in solution. Two-photon excitation is created when, by focusing an intensive light source, the density of photons per unit volume and per unit time becomes high enough for two photons to be absorbed into the same chromophore. In this case, the absorbed energy is the sum of the energies of the two photons. In two-photon excitation, dye molecules are excited only when both photons are absorbed simultaneously. The probability of absorption of two photons is equal to the product of probability distributions of absorption of the single photons. The emission of two photons is thus a quadratic process with respect to illumination intensity. Thus in two-photon excitation, only the fluorescence that is formed in the clearly restricted three-dimensional vicinity of the focal point is excited. We have developed an assay concept that is able to distinguish optically between the signal emitted from a microparticle in the focal point of the laser beam, and the signal emitted from the surrounding free labelled reagent. Moreover, the free labels outside the focal volume do not contribute any significant signal. This means that the assay is separation-free. The method based on two-photon fluorescence excitation makes possible fast single-step and separation-free immunoassays, for example, for whole blood samples. Since the method allows a separation-free assay in very small volumes, the method is very useful for high-throughput screening assays. Consequently we believe that two-photon fluorescence excitation will make a remarkable impact as a research tool and a routine method in many fields of analysis.

Background: The differential light-scattering pattern, an indicatrix, provides the most complete characterization of the optical properties of a particle. Particle classification can be performed on the basis of particle parameters retrieved from the indicatrices. This classification extends the ability of flow cytometry in particle recognition., Methods: The scanning flow cytometer (SFC) permits an acquisition of traces of light scattering signals, i.e., native SFC traces, from single particles. The acquired native SFC traces are transformed into indicatrices. The performance of the SFC in measurements of indicatrices has been demonstrated for the following particles: lymphocytes, erythrocytes, polystyrene particles, and milk-fat particles., Results: The structure and profile of the indicatrix for each particle type have been found to be unique. Classification of polystyrene particles has been performed on the basis of the map formed by particle refractive index and size. The polystyrene particles were classified using this map into different size categories ranging from 1.4-7 microm, with a size deviation of 0.07 microm., Conclusions: The method based on analysis of native SFC traces shows better performance in particle classification than the method based on the particle refractive index and size map. The classification performance of the SFC will be useful, for example, for particle sorting and particle identification, and with additional fluorescent measurements may have applications in multiparameter particle-based immunoassay., (Copyright 1999 Wiley-Liss, Inc.)

We studied the use of a dramatically reduced testing zone in combination with two-photon excitation and photon-burst analysis in high-throughput rare-event detection simulation using a modified flow cytometer. Two-photon excitation measurements were performed with a mode-locked titanium:sapphire laser. Fluorescence emission was measured with a photon-counting avalanche photodiode. Measured signal was analysed offline by autocorrelation and burst detection methods. Test samples were composed of full blood and orange fluorescent polystyrene nanospheres mixed in full blood. Results show that two-photon fluorescence excitation and time-correlation analysis provide a good signal-to-noise ratio for rare-event particle detection in a turbid sample environment.

We have studied the light-scattering properties of human erythrocytes both experimentally and theoretically. In the experimental study measurements were performed with a Scanning Flow Cytometer (SFC). The SFC can measure the light-scattering pattern (indicatrix) of an individual particle over an angular range of 10-60 degrees. We have observed polymorphism in the measured set of indicatrices. To understand the reason for this polymorphism, we have made a theoretical study of the scattering properties of erythrocytes. The Wentzel-Kramer-Brillouin approximation has been employed to calculate indicatrices of individual erythrocytes in different orientations relative to the incident light beam. The indicatrices were calculated over an angular range of 15-35 degrees. A comparison of the experimentally measured and theoretically calculated indicatrices shows that the polymorphism is due mainly to the different orientation of the erythrocytes in the flow. The effect caused by the Poiseuille profile of the flow on an individual erythrocyte within the SFC cuvette capillary was studied theoretically by use of the Stokes approximation. Rotation of an erythrocyte was predicted by this theoretical analysis, and this prediction was further verified by comparison with experimental results.

We introduce a new design for the optical cuvette and a new optical lay-out for the Scanning Flow Cytometer (SFC) that permits measurement of the angular dependency of the scattered light from individual moving particles. The improved optical scheme of the SFC allows measurement of the angular scattering pattern of individual particles at polar angles from 10 degrees to 120 degrees with integration at azimuthal angles from 0 degrees to 360 degrees and with angular resolution of better than 0.5 degrees. The performance of the SFC is demonstrated using certified polystyrene particles as reference material The aim of this work is to develop a flow cytometer, which, by recording the entire light scattering pattern of individual biological particles, would provide more information about the particle structure than the ordinary wide angle, forward and side scattering concepts.

We describe the three-dimensional (3-D) image formation and data acquisition in a stage scanning 4Pi confocal fluorescence microscope with the use of two-photon excitation. The 3-D point-spread functions of the 4Pi confocal and regular confocal microscope are measured and compared. Particular emphasis is given to the data acquisition procedure. 4Pi confocal microscopy results in a point-spread function that is 4 times sharper than that of a regular confocal microscope, ultimately leading to superior 3-D imaging of translucent fluorescent specimens. For a two-photon excitation wavelength of approximately 800 nm, we obtain an axial resolution of 140 nm.