Light Scatter Intensity Research Articles

Rapid and sensitive detection of miRNA via light scatter-aided emulsion-based isothermal amplification using a custom low-cost device

MicroRNAs are likely to be a next-generation clinical biomarker for many diseases. While gold-standard technologies, e.g., reverse transcription-quantitative polymerase chain reaction (RT-qPCR), exist for microRNA detection, there is a need for rapid and low-cost testing. Here, an emulsion loop-mediated isothermal amplification (eLAMP) assay was developed for miRNA that compartmentalizes a LAMP reaction and shortens the time-to-detection. The miRNA was a primer to facilitate the overall amplification rate of template DNA. Light scatter intensity decreased when the emulsion droplet got smaller during the ongoing amplification, which was utilized to moitor the amplification non-invasively. A custom low-cost device was designed and fabricated using a computer cooling fan, a Peltier heater, an LED, a photoresistor, and a temperature controller. It allowed more stable vortexing and accurate light scatter detection. Three miRNAs, miR-21, miR-16, and miR-192, were successfully detected using the custom device. Specifically, new template and primer sequences were developed for miR-16 and miR-192. Zeta potential measurements and microscopic observations confirmed emulsion size reduction and amplicon adsorption. The detection limit was 0.01 fM, corresponding to 2.4 copies per reaction, and the detection could be made in 5 min. Since the assays were rapid and both template and miRNA + template could eventually be amplified, we introduced the success rate (compared to the 95% confidence interval of the template result) as a new measure, which worked well with lower concentrations and inefficient amplifications. This assay brings us one step closer to allowing circulating miRNA biomarker detection to become commonplace in the clinical world.

Reliability of Aqueous Flare Measurements During Uveitis by a Spot Fluorometer

Purpose: To evaluate the reliability of intensity of light scatter (ILS) for diagnosing mild-to-moderate uveitis, which is difficult to score by the subjective standardized uveitis nomenclature (SUN) grading. Methods: ILS (in millivolts) was measured in healthy subjects and uveitis patients by a custom-made Spot fluorometer. The reliability was assessed by noise analysis of the instrument using turbid samples and intra- and interobserver variabilities. Results: ILS increased with the severity of inflammation: healthy (0.1498 ± 0.0564) and SUN0 (0.1590 ± 0.0564) < SUN1+ (0.2050 ± 0.0887) < SUN2+ (0.4283 ± 0.1895). The increase in ILS was more significant between healthy and SUN1+ (P < 0.0001) compared to the difference between SUN0 and SUN1+ (P = 0.0006). The instrument noise was small (σ = 0.002). Intraobserver measurements revealed no variability (P > 0.98) between measurements and predicted repeatability coefficients of 0.0754, 0.0522, and 0.0532 in healthy, SUN0, and SUN1+, respectively. Interobserver measurements also showed no variability (P > 0.88) and predicted intraclass correlation coefficient > 0.63 in healthy and uveitis subjects. Conclusions: The fluorometer enables precise measurement of ILS with negligible instrument noise, and the measurements are unaffected by intra- and interobserver variabilities. However, the difference in mean ILS between SUN0 and SUN1+ is greater than the instrument noise and comparable to intra- and interobserver variabilities. Thus, ILS measurements are likely impacted by blinking, eye movements, and focusing jitter. The impact of these factors must be reduced before using the fluorometer to distinguish mild-to-moderate uveitis.

Contamination-resistant, rapid emulsion-based isothermal nucleic acid amplification with Mie-scatter inspired light scatter analysis for bacterial identification

An emulsion loop-mediated isothermal amplification (eLAMP) platform was developed to reduce the impact that contamination has on assay performance. Ongoing LAMP reactions within the emulsion droplets cause a decrease in interfacial tension, causing a decrease in droplet size, which results in decreased light scatter intensity due to Mie theory. Light scatter intensity was monitored via spectrophotometers and fiber optic cables placed at 30° and 60°. Light scatter intensities collected at 3 min, 30° were able to statistically differentiate 103 and 106 CFU/µL initial Escherichia coli O157:H7 concentrations compared to NTC (0 CFU/µL), while the intensity at 60° were able to statistically differentiate 106 CFU/µL initial concentrations and NTC. Control experiments were conducted to validate nucleic acid detection versus bacterial adsorption, finding that the light scatter intensities change is due specifically to ongoing LAMP amplification. After inducing contamination of bulk LAMP reagents, specificity lowered to 0% with conventional LAMP, while the eLAMP platform showed 87.5% specificity. We have demonstrated the use of angle-dependent light scatter intensity as a means of real-time monitoring of an emulsion LAMP platform and fabricated a smartphone-based monitoring system that showed similar trends as spectrophotometer light scatter data, validating the technology for a field deployable platform.

Emulsion-based isothermal nucleic acid amplification for rapid SARS-CoV-2 detection via angle-dependent light scatter analysis

The SARS-CoV-2 pandemic, an ongoing global health crisis, has revealed the need for new technologies that integrate the sensitivity and specificity of RT-PCR tests with a faster time-to-detection. Here, an emulsion loop-mediated isothermal amplification (eLAMP) platform was developed to allow for the compartmentalization of LAMP reactions, leading to faster changes in emulsion characteristics, and thus lowering time-to-detection. Within these droplets, ongoing LAMP reactions lead to adsorption of amplicons to the water-oil interface, causing a decrease in interfacial tension, resulting in smaller emulsion diameters. Changes in emulsion diameter allow for the monitoring of the reaction by use of angle-dependent light scatter (based off Mie scatter theory). Mie scatter simulations confirmed that light scatter intensity is diameter-dependent and smaller colloids have lower intensity values compared to larger colloids. Via spectrophotometers and fiber optic cables placed at 30° and 60°, light scatter intensity was monitored. Scatter intensities collected at 5 min, 30° could statistically differentiate 10, 103, and 105 copies/μL initial concentrations compared to NTC. Similarly, 5 min scatter intensities collected at 60° could statistically differentiate 105 copies/μL initial concentrations in comparison to NTC. The use of both angles during the eLAMP assay allows for distinction between high and low initial target concentrations. The efficacy of a smartphone-based platform was also tested and had a similar limit of detection and assay time of less than 10 min. Furthermore, fluorescence-labeled primers were used to validate target nucleic acid amplification. Compared to existing LAMP assays for SARS-CoV-2 detection, these times-to-detections are very rapid.

Single-particle characterization of theranostic liposomes with stimulus sensing and controlled drug release properties

Stimuli-responsive nanotheranostic systems, integrated with diagnosis and treatment features, have recently emerged and attracted much interest. However, most of the research mainly focuses on the novelty of nanomaterials, and undervalues the significance of single-particle characterization which can provide detailed physical and biochemical information for performance evaluation and heterogeneity assessment. Due to the small particle size and low content of functional modules, high throughput and multiparameter analysis of individual stimuli-responsive nanoparticles still remains challenging. Here, fabricating a reactive oxygen species (ROS)-responsive liposome (Lipo@BODIPY11) and taking it as an example, we report the development of a strategy for theranostic nanoparticle characterization by a laboratory-built nano-flow cytometer (nFCM). Coincident detection of light scatter and fluorescence intensity provided a measure for liposome quality assessment. Theranostic performance referred to stimuli-responsive capability and drug release behavior upon ROS treatment were obtained in minutes. Besides, the dissociation of functional modules from liposomes and the formation of aggregates under high modification degree were revealed, which was otherwise masked by ensemble-averaged methods. At last, consistent results were also observed in intracellular studies. This nFCM-based method provides a comprehensive approach for the proof-of-principle study, heterogeneity assessment and quality control of biochemical nanosensors and theranostic nanomaterials.

Ocular Spot Fluorometer Equipped With a Lock-In Amplifier for Measurement of Aqueous Flare.

PurposeTo evaluate a custom-made ocular fluorometer for detection of intensity of light scatter (ILS) from the anterior chamber (A/C) as an objective measure of aqueous flare.MethodsThe fluorometer, equipped with a lock-in amplifier, was employed in the scatter mode to detect ILS from A/C. Measurements were performed with two illumination slit widths of 0.5 and 0.25 mm. The axial resolution at these slit widths were 80 and 200 μm, respectively. Healthy and pseudophakic eyes, with grade 0 Standardization of Uveitis Nomenclature (SUN) score, were employed as control subjects. ILS was also recorded in a cohort of patients who had undergone phacoemulsification and showed grades 1+ or 2+ on postoperative days 1 and 4.ResultsThe inter- and intraobserver variabilities in the measurement of ILS were not significant. In cataract patients, ILS was significantly higher on postoperative day 1 relative to healthy eyes. By day 4, ILS decreased significantly and was only marginally different from ILS in quiet pseudophakic eyes or healthy eyes. Eyes with higher SUN scores showed proportionately increased ILS. The receiver-operator characteristic analysis indicated no advantage in using the smaller slit width in discriminating ILS at different SUN scores although it provided higher axial resolution.ConclusionsThe lock-in–based spot fluorometer is reliable for measurement of ILS with high precision and accuracy.The measured ILS correlates linearly with SUN scores and can be used to provide a higher granularity for recording aqueous flare.Translational RelevanceThe instrument can be used in the clinical management of uveitis and drug development toward uveitis.

Concurrent detection of lysosome and tissue transglutaminase activation in relation to cell cycle position during apoptosis induced by different anticancer drugs.

Described is the new cytometric approach do detect either stimulation or a collapse of lysosomal proton pump (lysosomes rupture) combined with activation of transglutaminase 2 (TG2) during induction of apoptosis. Apoptosis of human lymphoblastoid TK6 cells was induced by combination of 2-deoxyglucose with the isoquinoline alkaloid berberine, by DNA topoisomerase I inhibitor camptothecin, its analog topotecan, topoisomerase II inhibitors etoposide or mitoxantrone, as well as by the cytotoxic anticancer ribonuclease ranpirnase (onconase). Activity of the proton pump of lysosomes was assessed by measuring entrapment and accumulation of the basic fluorochrome acridine orange (AO) resulting in its metachromatic red luminescence (F>640 ) within these organelles. Activation of TG2 was detected in the same cell subpopulation by the evidence of crosslinking of cytoplasmic proteins revealed by the increased intensity of the side light scatter (SSC) as well as following cell lysis by detergent, by its red fluorescence after staining by sulforhodamine 101. Because at low AO concentration nuclear DNA of the lysed cells was stoichiometrically stained green (F530 ) its quantity provided information on effects of the drug treatments on cell cycle in relation to activation of TG2. The data reveal that activation of lysosomal proton pump was evident in subpopulations of cells treated with 2-deoxyglucose plus berberine, topotecan, etoposide and mitoxantrone but not with ranpirnase. The collapse of lysosomal proton pump possibly reporting rupture of these organelles was observed in definite cell subpopulations after treatment with each of the studied drugs. Because regardless of the inducer of apoptosis TG2 activation invariably was correlated with lysosomes rupture it is likely that it was triggered by calcium ions or protons released from the ruptured lysosomes. This new methodological approach offers the means to investigate mechanisms and factors affecting autophagic lysosomes proton pump activity vis-à-vis TG2 activation that are common in several pathological states. © 2019 International Society for Advancement of Cytometry.

The Optical Integration technique for the measurement of light scatter in the human eye

AbstractPurpose To develop and demonstrate a new optical technique for the measurement of light scatter in the human eye based on extended source imaging in a double pass configuration.Methods The experimental setup is based on a double‐pass (DP) configuration. Series of disks of uniform radiance having angular dimensions ranging from 0 to 9.1 degrees (radius) are projected on to the eye's retina and DP images are recorded by a cooled electron‐multiplied CCD camera. Two spatially separated sub‐apertures at the pupil are used for stimulus projection and imaging to minimise back‐scatter and reflections. The derivative of the intensity of each recorded disk with respect to disk radius (in angular units) was fitted to a mathematical model of the wide‐angle double pass point‐spread function (PSF) and from that the underlying single‐pass PSF was computed. The setup was first validated using artificial eyes with known scattering properties and used in a group of normal human eyes.Results The derivative of central disk intensity with respect to disk radius was measurable up to the complete range of the angular dimensions of the projected disks (9.1 degrees of visual field). Different features can be obtained from the estimated PSF to characterize not only the amount of scatter but also its angular dependence.Conclusion A new optical method to measure scatter in the eye was developed and demonstrated. The procedure is sensitive enough to consistently detect differences in the light scatter intensity between normal healthy eyes and can be a significant step towards the complete optical characterization of the eye.

Direct and sensitive detection of foodborne pathogens within fresh produce samples using a field-deployable handheld device

Direct and sensitive detection of foodborne pathogens from fresh produce samples was accomplished using a handheld lab-on-a-chip device, requiring little to no sample processing and enrichment steps for a near-real-time detection and truly field-deployable device. The detection of Escherichia coli K12 and O157:H7 in iceberg lettuce was achieved utilizing optimized Mie light scatter parameters with a latex particle immunoagglutination assay. The system exhibited good sensitivity, with a limit of detection of 10CFUmL−1 and an assay time of <6min. Minimal pretreatment with no detrimental effects on assay sensitivity and reproducibility was accomplished with a simple and cost-effective KimWipes filter and disposable syringe. Mie simulations were used to determine the optimal parameters (particle size d, wavelength λ, and scatter angle θ) for the assay that maximize light scatter intensity of agglutinated latex microparticles and minimize light scatter intensity of the tissue fragments of iceberg lettuce, which were experimentally validated. This introduces a powerful method for detecting foodborne pathogens in fresh produce and other potential sample matrices. The integration of a multi-channel microfluidic chip allowed for differential detection of the agglutinated particles in the presence of the antigen, revealing a true field-deployable detection system with decreased assay time and improved robustness over comparable benchtop systems. Additionally, two sample preparation methods were evaluated through simulated field studies based on overall sensitivity, protocol complexity, and assay time. Preparation of the plant tissue sample by grinding resulted in a two-fold improvement in scatter intensity over washing, accompanied with a significant increase in assay time: ∼5min (grinding) versus ∼1min (washing). Specificity studies demonstrated binding of E. coli O157:H7 EDL933 to only O157:H7 antibody conjugated particles, with no cross-reactivity to K12. This suggests the adaptability of the system for use with a wide variety of pathogens, and the potential to detect in a variety of biological matrices with little to no sample pretreatment.

Usefulness of measuring reticulocyte hemoglobin equivalent in the management of haemodialysis patients with iron deficiency

The evaluation of iron status in dialysis patients provides information essential to the planning of adequate recombinant human erythropoietin treatment. The cellular iron status of the patients can be determined from the recently available measurement of reticulocyte hemoglobin equivalent (RET-He). RET-He is measured on the basis of automated fluorescent flow cytometry which in the reticulocyte channel, using a polymethine dye, also measures the mean value of the forward light scatter intensity of mature red blood cells and reticulocytes. These values equate with reticulocyte hemoglobin content. In this study, to clarify the accuracy of RET-He in diagnosing iron deficiency in dialysis patients, we initially compared RET-He with such iron parameters as serum ferritin levels, transferrin saturation and content of reticulocyte hemoglobin (CHr) which has been established as indicators of functional iron deficiency. Secondly, we investigated the changes in RET-He during iron supplementation for iron-deficient patients to determine whether this marker is a prospective and reliable indicator of iron sufficiency. The participants in this study were 217 haemodialysis patients. Iron deficiency was defined as havsing a transferrin saturation (TSAT) < 20% or serum ferritin < 100 ng/ml. Conventional parameters of red blood cells and RET-He were measured by on a XE-2100 automated blood cell counter (Sysmex). CHr was measured on an ADVIA120 autoanalyser (Siemens). RET-He mean value was 32.4 pg and good correlation (r = 0.858) between RET-He and CHr is obtained in dialysis patients. Receiver operating characteristic curve analysis revealed, values of the area was 0.776 and at a cutoff value of 33.0 pg, a sensitivity of 74.3% and a specificity of 64.9%, were achieved. Iron supplements given to the patients with low TSAT or ferritin, RET-He responded within 2 weeks, and this seemed to be a potential advantage of using RET-He in the estimation of iron status. RET-He is a new parameter, equivalent value to CHr, and is easily measurable on the widely spread and popular blood cell counter and is a sensitive and specific marker of iron status in dialysis patients.

Asymmetric division of Hansenula polymorpha reflected by a drop of light scatter intensity measured in batch microtiter plate cultivations at phosphate limitation

Hansenula polymorpha RB11 pC10-FMD (P(FMD)- GFP) (FMD promoter gfp gene) was simultaneously cultivated in the Respiration Activity Monitoring System (RAMOS) and in the microtiter plate cultivation system "BioLector" under phosphate limitation. The light scatter signal of the BioLector, for the determination of the biomass concentration in the wells, shows a significant decrease with the onset of the phosphate limitation until a stationary level is reached. At lower initial phosphate concentration this effect is more pronounced and longer time is required until the stationary level of the scattered light is achieved. The oxygen transfer rate signal of the RAMOS and the light scatter signal of the BioLector correlate with respect to the points of time where the maxima and the stationary levels of the courses are reached. In order to understand the effect causing this light scatter behavior, the forward and side scatter properties were investigated off line by flow cytometry. The decay in the light scatter of the BioLector seems to correlate with the formation of two subpopulations of different scatter intensities detected by a flow cytometer. With ongoing cultivation the fraction of cells possessing higher light scattering properties decreases until only a population of lower light scattering properties exists. The rate of transition of the yeast from one subpopulation to the other appears to be correlated with the rate of decrease in the BioLector light scatter signal. The formation of the subpopulations may be caused by an increased asymmetry in the cell cycle due to phosphate limitation.

A theory for viral capsid assembly around electrostatic cores

We develop equilibrium and kinetic theories that describe the assembly of viral capsid proteins on a charged central core, as seen in recent experiments in which brome mosaic virus capsids assemble around nanoparticles functionalized with polyelectrolyte. We model interactions between capsid proteins and nanoparticle surfaces as the interaction of polyelectrolyte brushes with opposite charge using the nonlinear Poisson Boltzmann equation. The models predict that there is a threshold density of functionalized charge, above which capsids efficiently assemble around nanoparticles, and that light scatter intensity increases rapidly at early times without the lag phase characteristic of empty capsid assembly. These predictions are consistent with and enable interpretation of preliminary experimental data. However, the models predict a stronger dependence of nanoparticle incorporation efficiency on functionalized charge density than measured in experiments and do not completely capture a logarithmic growth phase seen in experimental light scatter. These discrepancies may suggest the presence of metastable disordered states in the experimental system. In addition to discussing future experiments for nanoparticle-capsid systems, we discuss broader implications for understanding assembly around charged cores such as nucleic acids.

The Usefulness of CD71 Expression by Flow Cytometry for Differentiating Indolent From Aggressive CD10+ B-Cell Lymphomas

We studied 34 low- and 30 high-grade CD10+ B-cell lymphomas. Forward light scatter (FSC) and CD71 fluorescence intensity (CD71i) of tumor cells were measured and normalized by corresponding values for resting T cells. Significant differences in CD71i values between low- and high-grade lymphomas were observed by the Mann-Whitney U test (P < .001) and receiver operating characteristic (ROC) curve analysis (P < .001). FSC was not significantly different between low- and high-grade lymphomas; the area under the ROC curve was less than that for CD71i. Neither FSC nor CD71i significantly differentiated follicular lymphoma (FL) grades. A comparison of all FLs (grades 1-3) and non-FL high-grade lymphomas (Burkitt lymphoma [BL] and large B-cell lymphoma [LBCL]) showed significant differences in CD71i (P < .001) and FSC (P = .021). Differences were significant in CD71i and FSC between FL and LBCL (P < .001) but not between FL and BL. CD71i is more potent than FSC for distinguishing CD10+ low- from high-grade lymphomas and FL from non-FL high-grade lymphomas. Sensitivity and specificity are limited owing to inability to identify FL3. In ROC analysis, a high value for CD71i can identify BL and LBCL with a high degree of certainty.

The Usefulness of CD71 Expression by Flow Cytometry for Differentiating Indolent From Aggressive CD10+ B-Cell Lymphomas

We studied 34 low- and 30 high-grade CD10+ B-cell lymphomas. Forward light scatter (FSC) and CD71 fluorescence intensity (CD71i) of tumor cells were measured and normalized by corresponding values for resting T cells. Significant differences in CD71i values between low- and high-grade lymphomas were observed by the Mann-Whitney U test (P < .001) and receiver operating characteristic (ROC) curve analysis (P < .001). FSC was not significantly different between low- and high-grade lymphomas; the area under the ROC curve was less than that for CD71i. Neither FSC nor CD71i significantly differentiated follicular lymphoma (FL) grades. A comparison of all FLs (grades 1-3) and non-FL high-grade lymphomas (Burkitt lymphoma [BL] and large B-cell lymphoma [LBCL]) showed significant differences in CD71i (P < .001) and FSC (P = .021). Differences were significant in CD71i and FSC between FL and LBCL (P < .001) but not between FL and BL. CD71i is more potent than FSC for distinguishing CD10+ low- from high-grade lymphomas and FL from non-FL high-grade lymphomas. Sensitivity and specificity are limited owing to inability to identify FL3. In ROC analysis, a high value for CD71i can identify BL and LBCL with a high degree of certainty.

Whole blood fixation and permeabilization protocol with red blood cell lysis for flow cytometry of intracellular phosphorylated epitopes in leukocyte subpopulations

Previous studies of intracellular expression of phospho-epitopes in human leukocytes using flow cytometry have used erythrocyte removal or lysis before fixation. Because many of the phospho-epitopes of interest are part of signaling networks that respond to the environment and turn over rapidly, the interval and manipulations used to eliminate erythrocytes from samples have the potential to introduce artifacts. We report a procedure to fix samples containing red blood cells with formaldehyde and then remove erythrocytes by lysis. Detection of phospho-Thr 202/Tyr 204-p44/42 extracellular-regulated kinase (ERK) after phorbol ester acetate (PMA) stimulation was used as a model to measure phospho-epitopes in leukocyte populations in whole blood. Normal blood samples were activated with PMA followed by formaldehyde fixation and subsequent treatments with detergents and protein denaturants. The effects of each treatment were monitored by light scatter, selected CD expression intensity, and phosphorylated ERK (pERK) expression. Red cells could be lysed using 0.1% Triton X-100 after brief fixation of whole blood with 2% or 4% formaldehyde. Light scatter improved as a function of formaldehyde concentration and inversely with MeOH concentration. CD3 signal intensity increased when MeOH concentration was reduced. The ratio of pERK immunofluorescence in PMA-stimulated versus nonstimulated (control) samples was highest with high MeOH (90%) and lowest without MeOH treatment. This pattern is consistent with epitope unmasking by alcohol. The pERK epitope could also be unmasked by treatment with high salt, urea, acid, or heat, but none of these produced the level of unmasking of MeOH and each of these was associated with degradation of light scatter and CD3 staining intensity. The final procedure employed 4% formaldehyde, 0.1% Triton X-100, followed by 50% methanol denaturation. Samples prepared in this way demonstrated good preservation of light scatter and surface immunophenotypic patterns, similar to those obtained using a commercial whole blood/red blood cell lysing system (Q-Prep) and an acceptable PMA-stimulated pERK signal (essentially 100% of CD3+ cells that are pERK positive). Brief fixation of whole blood in 4% formaldehyde followed by treatment with Triton X-100 results in erythrocyte lysis and leukocyte light scatter and immunophenotypic features equivalent to those of other commercial lysis reagents. Intracellular pERK staining is significantly improved by treatment with methanol, but levels of MeOH above 50% degrade light scatter and CD3 expression. This protocol (formaldehyde/Triton X-100/MeOH) circumvents potential artifactual changes in phospho-epitopes due to removal of erythrocytes or erythrocyte lysis followed by fixation, and results in a pERK signal that resolves positive from negative cell populations.

Negligible direct radiative forcing of basin‐scale climate by coccolithophore blooms

The water‐leaving radiance, defined as radiation from the sun reflected off particles in water and exiting the ocean surface back into the atmosphere and space, is often used to derive ocean‐colour information from remotely sensed data. However, it is in itself a measure of the amount of solar irradiance reflected by oceanic constituents and, therefore, not available to the Earth's heat reservoir (changes in which can affect the Earth's energy balance and climate). A strong influence on the water‐leaving radiance is observed from coccolithophore blooms, owing to the highly reflective calcareous platelets or coccoliths covering these marine algae. We analysed remotely sensed water‐leaving radiances (1998–1999) over the N. Atlantic, where the blooms are spatially and temporally most abundant, and found that the direct radiative forcing of climate between 402–565 nm (the major range of optical influence) by coccolithophores in this ocean is negligible (∼0.05 W m−2 mean annually). This is in contrast to what in situ or laboratory measurements on the immense local intensity of coccolithophore visible light scatter in the past two decades have led us to believe.

Real-time observation of the change in light scattering from droplets with increasing deformity

Commercial aerodynamic particle sizing instruments generally achieve the desired particle size measurement by accelerating a sample airstream in which the particles are suspended and measuring the velocity acquired by individual particles. The accelerating flow regime can cause liquid droplets to deform and this subsequently introduces errors. In this paper, we present an apparatus that enables droplet deformation to be observed by recording the spatial light scatter intensity. The paper presents experimental data in video format showing the changes that occur in the light scattering from droplets as a function of increasing flow rate/deformation.

Monitoring phytoplankton, bacterioplankton, and virioplankton in a coastal inlet (Bedford Basin) by flow cytometry.

To establish the prevailing state of the ecosystem for the assessment of long-term change, the abundance of microbial plankton in Bedford Basin (Nova Scotia, Canada) is monitored weekly by flow cytometry. Phytoplankton are detected by their chlorophyll autofluorescence. Those that contain phycoerythrin are designated as Synechococcus cyanobacteria or cryptophyte algae according to the intensity of light scatter. Bacteria and viruses are stained with DNA-binding fluorochromes and detected by green fluorescence. Distinction is made between bacterial and viral subpopulations exhibiting high and low fluorescence. Time series data are presented for weekly observations from 1991 to 2000. Weekly averages are computed for the complete annual cycle of temperature, salinity, river discharge, nitrate, phosphate, silicate, chlorophyll, total phytoplankton including Synechococcus and cryptophytes, total bacteria including high and low-fluorescence subpopulations, and total viruses including high and low-fluorescence subpopulations. The microbial biomass in the surface water of Bedford Basin is dominated by phytoplankton. The spring bloom of phytoplankton represents a maximum in algal biovolume, but not in cell number. Phytoplankton, bacteria, and viruses all attain their annual numerical maxima between the summer solstice and the autumn equinox. A vigorous microbial loop and viral shunt is envisioned to occur in the summer.

Preparation, Microscopy, and Flow Cytometry with Excitation into Surface Plasmon Resonance Bands of Gold or Silver Nanoparticles on Aminodextran-Coated Polystyrene Beads

Spherical polystyrene latex beads of about 2.0 μm diameter were coated with islands of silver or gold metal, about 5−200 nm in diameter, by reduction of aqueous silver or gold ions in the presence of sugar-coated polystyrene latex beads. The metal islands are held on the bead surface by a polymeric sugar derivative, aminodextran, covalently bound to the polystyrene aldehyde/sulfate bead. Images of the gold or silver nanoparticle-coated polystyrene beads, obtained with an optical microscope, show that gold, silver, and uncoated polystyrene beads can be distinguished by their different colors, red-purple, black, and colorless, respectively. Also, scanning electron micrographs of coated versus uncoated polystyrene beads show a distinct granular bead surface when metal nanostructures are present versus a smooth bead surface when they are absent. Nanoparticles of gold greater than about 50 nm in diameter on polystyrene beads showed enhanced 90° or side light scatter (resonant Rayleigh scattering) with excitation at 633 nm but no enhancement (same light scatter intensity as uncoated polystyrene beads) at excitation wavelengths of 544, 488, and 458 nm. On the other hand, nanoparticles of silver greater than about 50 nm in diameter on polystyrene beads showed enhanced 90° or side light scatter with excitation wavelengths of 458, 488, 544, and 633 nm but no enhancement with 351−365 nm excitation. Amplification of elastic scatter from both silver and gold colloids was maximally achieved with structures of 50−200 nm diameter, as shown in forward versus side scatter histograms obtained by flow cytometry. Side scatter enhancements of 2- to 10-fold were observed for gold- or silver-coated polystyrene beads over uncoated polystyrene beads of the same diameter. The origin of this wavelength-dependent, light scattering amplification that was observed by flow cytometry has been identified with excitation into surface plasmon resonance bands of gold and silver nanoparticles on polystyrene latex beads.

3] Analysis of apoptotic cells by flow and laser scanning cytometry

A large number of flow cytometric methods to identify apoptotic cells and analyze morphological, biochemical, and molecular changes that occur during apoptosis have been developed. These methods are also applicable to the laser scanning cytometer (LSC), a microscope-based cytofluorometer that combines advantages of flow and image cytometry and that, by offering a possibility of assessment of cell morphology, is of particular utility in analysis of apoptosis. Apoptosis-related changes in cell morphology associated with cell shrinkage and condensation of cytoplasm and chromatin are detected by measurements of the intensity of light scatter of the laser beam in the forward and 90 degrees angle directions. Changes in plasma membrane composition and function are analyzed by its altered permeability to certain dyes and by the appearance of phosphatidylserine, which reacts with annexin V-fluorochrome conjugates on the external surface of the membrane. Decrease in mitochondrial transmembrane potential is measured with several fluorochromes of the rhodamine or carbocyanine family. DNA fragmentation is detected either by measurement of cellular DNA content after elution of the degraded DNA from the cell before or during the staining procedure or by in situ labeling DNA strand breaks. Apoptotic cells are then recognized either on the basis of their reduced DNA-associated fluorescence as the cells with fractional DNA content ("sub-G1 cells"), or as the cells with an extensive number of DNA breaks, respectively. Advantages and limitations of the preceding methods are discussed and their adaptation to LSC is presented.