Laser Fluorescence Detection Research Articles

Light sheet imaging and interactive analysis of the cardiac structure in neonatal mice.

Light-sheet microscopy (LSM) enables us to strengthen the understanding of cardiac development, injury, and regeneration in mammalian models. This emerging technique decouples laser illumination and fluorescence detection to investigate cardiac micro-structure and function with a high spatial resolution while minimizing photodamage and maximizing penetration depth. To unravel the potential of volumetric imaging in cardiac development and repair, we sought to integrate our in-house LSM, Adipo-Clear, and virtual reality (VR) with neonatal mouse hearts. We demonstrate the use of Adipo-Clear to render mouse hearts transparent, the development of our in-house LSM to capture the myocardial architecture within the intact heart, and the integration of VR to explore, measure, and assess regions of interests in an interactive manner. Collectively, we have established an innovative and holistic strategy for image acquisition and interpretation, providing an entry point to assess myocardial micro-architecture throughout the entire mammalian heart for the understanding of cardiac morphogenesis.

Effect of nanomicelle curcumin-based photodynamic therapy on the dynamics of white spot lesions and virulence of Streptococcus mutans in patients undergoing fixed orthodontic treatment: A randomized double-blind clinical trial

Background/purposeThe formation of white spot lesions (WSLs) around fixed orthodontic appliances is a major complication during treatment. The current double-blind, randomized clinical trial (RCT) study aims to investigate the varying effects of nanomicelle curcumin-based photodynamic therapy (NMCur-aPDT) on microbial count and virulence of Streptococcus mutans as well as the number and dynamics of WSLs. Materials and methodsDouble-blind prospective RCT, comprised of 48 patients with fixed orthodontic appliances, were recruited for the current study. The patients were divided into four groups according to the type of the treatment (NMCur, LED, NMCur-aPDT or VITIS® anti-caries mouthwash), using block randomization. Antimicrobial and anti-virulence activities of the treatments against isolated S. mutans were assessed via colony counting and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. The visual inspection using the International Caries Detection and Assessment System (ICDAS II) score and laser fluorescence (LF) detection using a DIAGNOdent device were used for the detection and assessment of the dynamics of WSLs, respectively, on the labial surface in four areas (i.e., gingival, incisal, mesial, and distal) of the upper and lower anterior teeth at 30-, 60-, 90-, and 120-days follow-up after bonding of the lower and upper arches. ResultsThe antimicrobial properties of NMCur, VITIS®, and NMCur-aPDT were time-dependent so the highest reduction in S. mutans population was observed following NMCur-aPDT (99.98%) on day 120 of the study. The gtfB gene expression levels in S. mutans isolates from the NMCur-aPDT group on days 60, 90, and 120 decreased by 2.07-, 2.32-, and 3.01-fold more than in S. mutans isolates from the VITIS® group, respectively (all P < 0.05), while NMCur and LED treatments could not significantly reduce gtfB gene expression up to 120 days of follow-up (P > 0.05). In patients who were treated with LED, an increase in the mean number of WSLs per patient (mean increase, 1.8; P < 0.05) was found, while in NMCur-aPDT and VITIS® groups, not only no increases were observed, but the mean number of WSLs per patient decreased (mean reductions, 0.5 and 0.9, respectively; not significant). LED treatment caused significant increases (P < 0.05) in the mean LF values at 90-and 120-days of follow-up in comparison with the baseline (mean increases, 5.1 and 6.5, respectively) while, in NMCur-aPDT, VITIS®, and NMCur groups 11.8-, 7.1-, and 4.4-reductions in the mean LF values were observed, respectively (all, P < 0.05). ConclusionsThe antimicrobial and anti-virulence activities of NMCur-aPDT against S. mutans were higher than the other treatment groups. In patients who were treated with NMCur-aPDT, the mean number and LF values of WSLs per patient were significantly lower than the other groups in 90-and 120-days of follow-up.

Investigation of the chemical effect of pilot injection on main combustion in a gasoline controlled auto-ignition engine by in-cylinder measurements and numerical simulation of H2O2, HO2, and OH radicals

Compared to conventional combustion in gasoline engines, Gasoline Controlled Auto-Ignition (GCAI) has the potential to significantly reduce both fuel consumption and nitrogen oxides (NOX) raw emissions. However, GCAI combustion still has a limited operating range due to limitations in combustion stability at low loads and high rates of cylinder pressure rise at high engine loads. Previous research identified hydrogen peroxide (H2O2) decomposition as an effective means to control GCAI, which is formed among other species with pilot fuel injection during the negative valve overlap (NVO) phase. Since the chemical effect of H2O2 on the main combustion has not been experimentally confirmed so far, the aim of the experimental study described in this paper is to detect H2O2 during early compression using optical methods. This is approached by conducting Photofragmentation Laser-Induced Fluorescence (PFLIF) of H2O2 by a pump laser and simultaneous hydroxyl radical Laser-Induced Fluorescence (OH-LIF) detection in the combustion chamber of a dedicated GCAI single-cylinder research engine at low loads after NVO with pilot injection (PI). Thereto, the pilot injection strategy as well as the global air/fuel ratio (AFR) are varied. Interestingly, no H2O2, hydroxyl (OH) or hydroperoxyl (HO2) constituents were detected under the operating conditions studied, despite relatively high pilot injection fuel masses and a highly accurate H2O2 detection limit of about 1 ppm. Numerical three-dimensional computational fluid dynamics (3D-CFD) combustion simulations using a semi-detailed reaction kinetic mechanism predicted 5–8 times higher H2O2 concentrations than the detection limit, yielding an uncertainty estimate of the numerical methods. Furthermore, the 3D-CFD simulations showed a significant mixture stratification which is a possible reason for the lack of H2O2 detection under these operating conditions.

Inflow nano-optics from the near-to the far-field detection based on Metal-Enhanced Fluorescence signaling

In this communication we evaluated the detection and tracking of Ultraluminescent gold core–shell nanoplatforms based on Metal-Enhanced Fluorescence (MEF) within colloidal dispersion by In-Flow Cytometry and Imaging Flow Cytometry (IFC). The enhanced properties were developed by the incorporation of Rhodamine B (RhB) as a laser dye reporter at variable controlled silica spacer lengths (Au@SiO2-RhB) from a gold core template. Thus, SiO2 was varied from 6 to 7 nm to 25 nm in order to place the laser dye sensitizer at different lengths of the higher electromagnetic fields surrounding the 45-nm gold core diameter. By Laser Fluorescence Microscopy, high intense emissions were shown at shorter SiO2 lengths, while lower emissions were found towards longer ones. These characteristics related to MEF were evaluated by in-flow cytometry with dual laser excitation and fluorescence detection, and by an IFC set-up. By In-flow cytometry, different scattered-light distributions were shown, depending on the nanoparticle concentrations in the colloidal dispersion and size of the Au@SiO2-RhB. Moreover, varied fluorescent event detections and counting were recorded according to the SiO2 lengths applied. A ratio of fluorescent event detection counting of core–shell/core-less of 40–42 was established for optimal MEF Enhancement Factors (MEFEF) of 41–43 determined by Laser Fluorescence Microscopy. Core-less nanoparticles showed a marked decrease of fluorescent event detection counting caused by higher photobleaching and absence of the MEF effect. Hence, an improved detection effect was shown within in-flow based on MEF from single nanoplatforms. Therefore, the Ultraluminescence emission of core–shell nanoarchitectures within in-flow techniques was shown and discussed. A non-classical light in real time within colloidal gold core–shell nanoparticles was generated by accurate control of their architectures.

In flow metal-enhanced fluorescence for biolabelling and biodetection.

Escherichia coli bacteria were determined by in flow cytometry with laser excitation and fluorescence detection applying ultraluminescent core-shell nanoparticles based on Metal Enhanced Fluorescence (MEF). Core-shell nanoparticles consisted of a 40 nm core modified with a silica spacer grafted with Rhodamine B (RhB). The electromagnetic field in the near field of the core surface enhanced the fluorescence of RhB by plasmonic and fluorophore coupling. The hydrophilic silica spacer allowed the non-covalent interaction with the polar E. coli surface and thus ultraluminescent bacteria biolabelling was developed. Clearly, well defined and bright bacteria imaging was recorded by Laser Fluorescence Microscopy based on the non-covalent deposition of the ultraluminescent nano-emitters. Using these nano-labellers, it was possible to detect labelled E. coli by in flow cytometry. Higher values of Side-scattered light (SSC) and Forward-scattered light (FSC), and number of fluorescent event detections, were observed for labelled bacteria compared to those non-labelled. The sensitivity of the methodology was evaluated by varying bacteria concentration and acceptable analytical figures of merit were determined. Applying this methodology we could quantify E. coli from a synthetic real sample of fortified water. Similar results were obtained by bacteria counting with Laser Fluorescence Microscopy and with a cell-bacteria counter.

Compatibility of CO2 laser surgery and fluorescence detection in head and neck cancer cells.

Surgical treatment of cancer requires tumor excision with emphasis on function preservation which is achieved in (early stage) laryngeal cancer by transoral carbon dioxide (CO2 ) laser surgery. Whereas conventional laser surgery is restricted by the surgeon's visual recognition of tumor tissue, new approaches based on fluorescence-guided surgery (FGS) improve the detection of the tumor and its margin. However, it is unclear whether fluorophores are compatible with high-power laser application or whether precision is compromised by laser-induced bleaching of the dye. We applied topology-controlled 3D laser resection of fluorescent tumors cell in vitro and laser-induced autofluorescence analysis ex vivo. Laser-induced bleaching of fluorescent dyes in the visible and near-infrared light spectrum (650-900 nm) ranges below the resolution range of operation microscopes. Furthermore, specific fluorescent signals in an FGS mouse model is 104 higher than laser-induced autofluorescence in mouse tissue. Laser-induced lateral photobleaching is negligible indicating a path forward for fluorescence-guided laser surgery in head and neck cancer.

Quantification of multiple human immune checkpoint molecules using antibody array technology

Abstract Immune checkpoint molecules play an important role in T cell functionality after TCR/MHC signaling. Blockade of two B7/CD28 family checkpoint molecules, CTLA-4 and PD-1 have already demonstrated superior efficacies in treatment for a variety of malignancies. Recently, the US FDA approved one anti-CTLA-4 monoclonal antibody (ipilimumab, 2011) and two anti-PD-1 monoclonal antibodies (pembrolizumab 2014, and nivolumab 2014) for the treatment of advanced melanoma. Other immune checkpoint molecules have been increasingly considered as new targets for cancer immunotherapy. To facilitate better identification of novel targets and elucidation of the checkpoint blockade, simultaneous detection of these pathway molecules in complex biological samples is essential. With antibody array technology, a novel checkpoint molecule array was developed for quantitative measurement 10 of the most important B7/CD28 family checkpoint molecules including B7-1/CD80, B7-2/CD86, B7-H1/PD-L1, B7-H2/ICOSL, B7-H3, CD28, CTLA-4, ICOS, PD-1, and PD-L2/B7-DC. Antigen-specific capture antibodies were fixed on glass slides with a non-contact arrayer, and multiplex ELISA detection of these proteins was achieved through laser fluorescence detection. All the markers could be detected at pg/ml level. The array was validated by cross reactivity and media recovery tests, and was further validated by conventional ELISA testing of normal human sera. In summary, our system provides a powerful, efficient, and low-cost tool for immunology research and biomarker discovery.

Dye-enhanced laser fluorescence detection on natural caries lesions in primary teeth

Objective: This study aimed to investigate the association of two fluorescent dyes and Laser Fluorescence (LF) device in detecting smooth and occlusal natural caries in primary teeth in vitro.Methods: Measurements were performed with the LF and with LF associated with tetrakis (N-methylpyridyl)porphyrin (LF TMPyP) and protoporphyrin IX (LF PPIX) in 72 smooth (63 primary molars) and 134 occlusal sites (81 primary molars). For validation, surfaces were sectioned and sections obtained were evaluated under stereomicroscope. Smooth surfaces were also evaluated using polarized light microscopy and Knoop microhardness. For both smooth and occlusal surfaces, ROC analyses were performed, and sensitivities, specificities and accuracies were assessed. In smooth surfaces, Pearson’s correlation coefficients between LF values and lesions hardness or lesions depth were calculated.Results: LF TMPyP presented higher correlation with hardness and lesion depth than other methods in smooth surfaces. No differences were observed in other parameters among the methods, in both smooth and occlusal surfaces.Relevance: The LF TMPyP might improve performance in quantifying smooth-surface caries lesions in primary teeth. However, the sensitivity is improved at D2 (caries extending into inner half of the enamel but not to amelodentinal junction) threshold when using PPIX in smooth caries lesions. The association of LF with fluorescent dyes does not improve the performance on occlusal caries.

Detecting Proximal Secondary Caries Lesions

When choosing detection methods for secondary caries lesions, dentists need to weigh sensitivity, allowing early initiation of retreatments to avoid lesion progression, against specificity, aiming to reduce risks of false-positive diagnoses and invasive overtreatments. We assessed the cost-effectiveness of different detection methods for proximal secondary lesions using Monte Carlo microsimulations. A vital permanent molar with an occlusal-proximal restoration was simulated over the lifetime of an initially 20-y-old. Three methods were compared: biannual tactile detection, radiographic detection every 2 y, and biannual laser fluorescence detection. Methods were employed either on their own or in pairwise combinations at sensitive and specific thresholds estimated with systematically collected data. A mixed public-private payer perspective in the context of German health care was applied. Effectiveness was calculated as years of tooth retention. Net-benefit analyses were used to evaluate cost-effectiveness acceptability at different willingness-to-pay thresholds. Radiographic detection verified by tactile assessment (both at specific thresholds) was least costly (mean, 1,060 euros) but had limited effectiveness (mean retention time, 50 y). The most effective but also more costly combination was laser fluorescence detection verified by radiography, again at specific thresholds (1157 euros, 53 y, acceptable if willingness to pay >32 euro/y). In the majority of simulations, not combining detection methods or applying them at sensitive thresholds was less effective and more costly. Net benefits were not greatly altered by applying different discounting rates or using different baseline prevalence of secondary lesions. Current detection methods for secondary lesions should best be used in combination, not on their own, at specific thresholds to avoid false-positive diagnoses leading to costly and invasive overtreatment. The relevant characteristics, such as predictive value, of different methods should be assessed in longitudinal clinical studies.

Quantification of 500 human protein biomarkers using antibody arrays and its applications in immunology research (TECH2P.921)

Abstract Proteomic approach continues to be the trend for biomarker discovery through profiling as many proteins as possible in a biological sample. Though LC-MS technology can accurately detect medium-to-high abundance proteins (&amp;gt; ng/ml), many cellular modulators such as cytokines are present at pg/ml level. Immunoassays continue to be the most utilized tools for biomarker discovery and validation due to their convenience, sensitivity and specificity. Here we validated the sandwich detection of 500 most analyzed human protein biomarkers with a pair of antigen-specific antibodies. Subsequently, capture antibodies were fixed on glass slides with non-contact arrayer, and multiplex ELISA detection of these protein biomarkers was achieved through planar antibody array technology with laser fluorescence detection. To overcome the potential cross reactivity among different reagents, multiple non-overlapping arrays were developed based upon their cellular abundance, functionality, and cross reactivity. Most of the markers can achieve pg/ml detection - similar to single ELISA detection but with much wider dynamic range. These arrays were further validated with normal human sera, where over two thirds of these proteins are within the detection range. Separate array panels were developed for those proteins with high abundance in sera, where high sample dilutions are needed. In summary, our system provides a powerful, efficient, and low-cost tool for immunology research and biomarker discovery.

Laser fluorescence detection method of plant stress conditions caused by insufficient nutrients or contaminants in soil

Laser fluorescence detection method of plant stress conditions caused by insufficient nutrients or contaminants in soil

Automatic laser scanning ablation system for high-precision treatment of brain tumors

Complete removal of malignant gliomas is important for the prognosis in neurosurgery treatment. Currently, the challenge is how to detect any remaining tumors and resect them during the operation. We have developed a laser ablation system with accurate tumor analysis and fluorescence guidance for high-precision brain tumor resection during neurosurgery. A 5-aminolevulinic acid-induced fluorescent protoporphyrins IX (PpIX)-based intra-operative fluorescence measurement and corresponding spectra analysis technique is used to identify the position of tumors. A galvano mirror scanning mechanism is integrated into the fluorescence measurement and the laser ablation devices for automatic tumor area scanning and corresponding laser ablation. A set of phantom experiments was performed to evaluate the proposed system. Results showed that the galvano scanning mechanism enabled both PpIX fluorescence detection and laser ablation in the same optical axis. In vitro experiments using porcine brain were performed to evaluate the effectiveness of the automatic laser scanning, fluorescence detection, and laser ablation system. The proposed fluorescence-guided laser ablation system can provide accurate analysis and high-precision treatment for tumor resection in neurosurgery. With further improvement, the system can be used in neurosurgical implementation to provide accurate, safe, and simple surgical diagnosis and therapy.

Investigation of the hyperfine structure of lanthanum lines by a laser-induced fluorescence technique

Hyperfine constants of lanthanum I and II levels are reported, determined using laser excitation and fluorescence detection. Free La atoms and ions were produced in a hollow cathode discharge and excited using laser light, generated by a tunable ring dye laser, working with the dyes Coumarin 102, Coumarin 30 and Rhodamine 700. More than 150 spectral lines are investigated for the determination of the magnetic dipole coupling constants A and the electric quadrupole coupling constants B of 99 levels of La I and 28 levels of La II. The constants A and B for seven known levels of La I are reported for the first time in this work. Four new levels of La I are also discovered and confirmed by a second laser excitation.

Study of the Micro- and Nanostructured Silicon for Biosensing and Medical Applications

Emerging applications of porous silicon (PS) lies in its ability to incorporate other materials, such as organic groups, organic and inorganic nanoparticles to form (bio)hybrid systems where each individual constituent may be optimized for a particular function. This paper presents our recent experimental results on the fabrication and applications in biosensing of the porous silicon (PS) based microstructures. We have demonstrated that different morphologies of PS, either as-prepared or coated with gold nanoparticles have an important role in biomolecule detection, due to its large internal surface combined with specific electro-optical properties, being in the same time support for immobilization of complementary biomolecules as well as transducer for biochemical interactions. Therefore, we have investigated the photoluminescence properties of nanoporous Si prepared on different Si micropatterned surfaces comparatively with PS/flat Si in order to develop a new simple and versatile process for biosensor transducer fabrication. Meso- and macro-PS have been investigated for protein immobilization and detection using microarray technique or for DNA biomolecule detection by impedance spectroscopy. Finally, we have demonstrated that macroporous silicon constitutes an appropriate substrate for very sensitive SERS biosensors. RAMAN signal of 11-mercaptoundecanoic acid was investigated on Au/macroporous silicon. Various characterisation techniques have been used, optical and scanning electron microscopy (SEM) to investigate samples morphology, X-ray diffraction for nanoparticle structure, Raman and PL spectroscopy, and laser fluorescence detection for chemical and optical properties analysis and impedance spectroscopy for investigation organic molecule attachment on the Au/PS structures.

Dye-enhanced laser fluorescence detection of caries lesions around brackets

The aim was to evaluate the performance of DIAGNOdent [laser fluorescence(LF) and LFpen] devices enhanced by fluorescent dye in detecting mineral loss around brackets and comparing the inhibitory effect of bonding material on artificial demineralization, and to verify whether LF methods show the same trends of mineral loss. Brackets were bonded to premolar halves with Fuji Ortho LC, Transbond XT, and Ortho Glass LC cements (n = 15). The teeth were soaked in demineralizing solution (pH = 4.8) for 16 days. Mineral loss was calculated by atomic emission spectrometry, and lesions were measured with LF devices with dye [tetrakis N-methylpyridyl porphyrin (TMPyP)]. Groups were compared with regard to LF readings and mineral loss, and performance of caries detection was calculated. Higher mineral loss and LF-TMPyP values occurred in the resin group. LFpen-TMPyP readings were significantly higher in the demineralized groups. Correlation was observed between mineral loss and LF measurements. LF methods are capable of identifying lower demineralization around brackets bonded with resin-modified glass ionomer cements.

Absorption of NO laser-induced fluorescence by hot O 2 and CO 2

Compared to conventional combustion in gasoline engines, Gasoline Controlled Auto-Ignition (GCAI) has the potential to significantly reduce both fuel consumption and nitrogen oxides (NOX) raw emissions. However, GCAI combustion still has a limited operating range due to limitations in combustion stability at low loads and high rates of cylinder pressure rise at high engine loads. Previous research identified hydrogen peroxide (H2O2) decomposition as an effective means to control GCAI, which is formed among other species with pilot fuel injection during the negative valve overlap (NVO) phase. Since the chemical effect of H2O2 on the main combustion has not been experimentally confirmed so far, the aim of the experimental study described in this paper is to detect H2O2 during early compression using optical methods. This is approached by conducting Photofragmentation Laser-Induced Fluorescence (PFLIF) of H2O2 by a pump laser and simultaneous hydroxyl radical Laser-Induced Fluorescence (OH-LIF) detection in the combustion chamber of a dedicated GCAI single-cylinder research engine at low loads after NVO with pilot injection (PI). Thereto, the pilot injection strategy as well as the global air/fuel ratio (AFR) are varied. Interestingly, no H2O2, hydroxyl (OH) or hydroperoxyl (HO2) constituents were detected under the operating conditions studied, despite relatively high pilot injection fuel masses and a highly accurate H2O2 detection limit of about 1 ppm. Numerical three-dimensional computational fluid dynamics (3D-CFD) combustion simulations using a semi-detailed reaction kinetic mechanism predicted 5–8 times higher H2O2 concentrations than the detection limit, yielding an uncertainty estimate of the numerical methods. Furthermore, the 3D-CFD simulations showed a significant mixture stratification which is a possible reason for the lack of H2O2 detection under these operating conditions.

Indocyanine Green Fluorescence Measurement of Intestinal Transit and Gut Perfusion after Intestinal Manipulation

Background and Aims: Postoperative ileus is a common and poorly understood problem of abdominal surgery. The aim of this study was to measure postoperative intestinal transit and to evaluate bowel wall perfusion by a novel in vivo indocyanine green (ICG)-fluorescence measurement following intestinal manipulation (IM). Methods: Rats underwent a simple intestinal manipulation. Myeloperoxidase-positive cells in the muscularis were stained with the Hanker-Yates reaction and quantified histochemically. Bowel wall perfusion was determined directly and 24 h postoperatively using a laser-fluorescence detection unit. Intestinal transit was visualized 24 h after IM. Results: IM resulted in a massive infiltration (155-fold) of neutrophils into the intestinal muscularis 24 h postoperatively. Bowel wall perfusion significantly decreased directly and 24 h following surgery (29 and 59%, respectively). Gastrointestinal transit was similarly impaired and showed a reduction to 40% of the control values 24 h after IM. Conclusion: IM of the rat small intestine caused an impairment in bowel wall perfusion and microcirculation and a significant decrease in gastrointestinal transit. The ICG fluorescence measurement using the described system proved to be a simple and reliable method to evaluate intestinal transit and bowel wall microcirculation in vivo.

Integrated portable genetic analysis microsystem for pathogen/infectious disease detection.

An integrated portable genetic analysis microsystem including PCR amplification and capillary electrophoretic (CE) analysis coupled with a compact instrument for electrical control and laser-excited fluorescence detection has been developed. The microdevice contains microfabricated heaters, temperature sensors, and membrane valves to provide controlled sample positioning and immobilization in 200-nL PCR chambers. The instrument incorporates a solid-state laser and confocal fluorescence detection optics, electronics for sensing and powering the PCR reactor, and high-voltage power supplies for conducting CE separations. The fluorescein-labeled PCR products are amplified and electrophoretically analyzed in a gel-filled microchannel in <10 min. We demonstrate the utility of this instrument by performing pathogen detection and genotyping directly from whole Escherichia coli and Staphylococcus aureus cells. The E. coli detection assay consists of a triplex PCR amplification targeting genes that encode 16S ribosomal RNA, the fliC flagellar antigen, and the sltI shigatoxin. Serial dilution demonstrates a limit of detection of 2-3 bacterial cells. The S. aureus assay uses a femA marker to identify cells as S. aureus and a mecA marker to probe for methicillin resistance. This integrated portable genomic analysis microsystem demonstrates the feasibility of performing rapid high-quality detection of pathogens and their antimicrobial drug resistance.

PDMS-based opto-fluidic micro flow cytometer with two-color, multi-angle fluorescence detection capability using PIN photodiodes

This research describes the design and performance of a microfluidic observation channel capable of performing flow cytometry measurements using solid-state lasers and PIN-based photodetectors. The observation cell, fabricated using poly(dimethylsiloxane) (PDMS)-based soft lithography, is designed with integrated microgrooves that permit the physical registration of optical fibers that serve as optical waveguides for laser excitation and fluorescence detection. The optical fiber arrangement is designed to permit simultaneous multi-color excitation of sample particles, at a single interrogation point in the observation channel, using multiple angles of excitation and detection. The multiple angles can be used to increase signal-to-noise ratio ( S/ N) by performing two independent fluorescence measurements simultaneously, and to permit multiple simultaneous cytometric measurements to occur at a single interrogation point. Moreover, the multi-angle micro flow cytometer is proven to provide consistent color differentiation that allows the design to achieve spectrometer capabilities not possible on a conventional flow cytometer. The micro flow cytometer performance was evaluated by considering the detection of fluorescent microspheres (15 μm) and nucleic acid labeled fungus ( Saccharomyces cerevisiae). Multiple angles (45°, 135°, and 180°) between fluorescence excitation and detection were investigated with respect to S/ N, S/ N coefficient of variation, and particle recovery. The experiments demonstrate the feasibility of developing a high S/ N single-cell fluorescence detection system based exclusively on silicon-based PIN photodiodes coupled with the lock-in amplification technique. The technique is applicable to the detection of scatter and fluorescent signals emanating from inorganic particles and biological cells, and is highly amenable to economical manufacturing, miniaturization, and low-power operation. Consequently, the design approach and performance results described in this investigation have applicability to the development of practical and low-cost cytometric micro total analysis systems (μTAS).

Microarrays for genotyping human group a rotavirus by multiplex capture and type-specific primer extension.

Human group A rotavirus (HRV) is the major cause of severe gastroenteritis in infants worldwide. HRV shares the feature of a high degree of genetic diversity with many other RNA viruses, and therefore, genotyping of this organism is more complicated than genotyping of more stable DNA viruses. We describe a novel microarray-based method that allows high-throughput genotyping of RNA viruses with a high degree of polymorphism by multiplex capture and type-specific extension on microarrays. Denatured reverse transcription (RT)-PCR products derived from two outer capsid genes of clinical isolates of HRV were hybridized to immobilized capture oligonucleotides representing the most commonly occurring P and G genotypes on a microarray. Specific primer extension of the type-specific capture oligonucleotides was applied to incorporate the fluorescent nucleotide analogue cyanine 5-labeled dUTP as a detectable label. Laser scanning and fluorescence detection of the microarrays was followed by visual or computer-assisted interpretation of the fluorescence patterns generated on the microarrays. Initially, the method detected HRV in all 40 samples and correctly determined both the G and the P genotypes of 35 of the 40 strains analyzed. After modification by inclusion of additional capture oligonucleotides specific for the initially unassigned genotypes, all genotypes could be correctly defined. The results of genotyping with the microarray fully agreed with the results obtained by nucleotide sequence analysis and sequence-specific multiplex RT-PCR. Owing to its robustness, simplicity, and general utility, the microarray-based method may gain wide applicability for the genotyping of microorganisms, including highly variable RNA and DNA viruses.