Fluorescent Particle Concentrations Research Articles

Ambient bioaerosol particle dynamics observed during haze and sunny days in Beijing

The chemical characteristics of airborne particulate matter (PM) have been extensively studied; however, little information exists for its biological components (bioaerosol) especially during a haze event in mega cities. Herein, we studied the bioaerosol (fluorescent particle) dynamics on both haze and sunny days in Beijing from Dec. 2013 to March 2014 by employing a widely used real-time bioaerosol sensor-ultraviolet aerodynamic particle spectrometer (UV-APS). Firstly, we studied the fluorescent particle (BioPM) concentration and size distributions during three independent haze and three independent sunny days. Secondly, we investigated BioPM dynamics over a two-week long monitoring period which included consecutive haze days and alternated sunny days. In addition, we analyzed bacterial community structures and endotoxin levels in the air samples using pyrosequencing and Limulus amebocyte lysate (LAL) method, respectively.More than 6-fold higher fluorescent particle concentrations up to 5×105/m3 with peaks at night or early dawn were detected at the time of haze occurrences than those observed on sunny days. When the haze episode progressed for 3–5days, the BioPM concentrations were observed to decrease to the levels that were typically observed on sunny days. In general, ozone levels were found to be elevated at noon, while BioPM, NOx and relative humidity were reduced. Gene sequence analysis revealed no significant difference in abundances and community structures for top 13 bacterial genera between haze and sunny days, yet about twice higher endotoxin levels (12.4EU/m3) were detected on haze days than on sunny days. The results here facilitate a better understanding of atmospheric fluorescent particle dynamics including those under haze events.

Spatial Intensity Distribution Analysis Reveals Abnormal Oligomerization of Proteins in Single Cells

Knowledge of membrane receptor organization is essential for understanding the initial steps in cell signaling and trafficking mechanisms, but quantitative analysis of receptor interactions at the single-cell level and in different cellular compartments has remained highly challenging. To achieve this, we apply a quantitative image analysis technique—spatial intensity distribution analysis (SpIDA)—that can measure fluorescent particle concentrations and oligomerization states within different subcellular compartments in live cells. An important technical challenge faced by fluorescence microscopy-based measurement of oligomerization is the fidelity of receptor labeling. In practice, imperfect labeling biases the distribution of oligomeric states measured within an aggregated system. We extend SpIDA to enable analysis of high-order oligomers from fluorescence microscopy images, by including a probability weighted correction algorithm for nonemitting labels. We demonstrated that this fraction of nonemitting probes could be estimated in single cells using SpIDA measurements on model systems with known oligomerization state. Previously, this artifact was measured using single-step photobleaching. This approach was validated using computer-simulated data and the imperfect labeling was quantified in cells with ion channels of known oligomer subunit count. It was then applied to quantify the oligomerization states in different cell compartments of the proteolipid protein (PLP) expressed in COS-7 cells. Expression of a mutant PLP linked to impaired trafficking resulted in the detection of PLP tetramers that persist in the endoplasmic reticulum, while no difference was measured at the membrane between the distributions of wild-type and mutated PLPs. Our results demonstrate that SpIDA allows measurement of protein oligomerization in different compartments of intact cells, even when fractional mislabeling occurs as well as photobleaching during the imaging process, and reveals insights into the mechanism underlying impaired trafficking of PLP.

Scanning fluorescence correlation spectroscopy on biomembranes.

Fluorescence correlation spectroscopy (FCS) is a powerful quantitative method to study dynamical properties of biophysical systems. It exploits the temporal autocorrelation of fluorescence intensity fluctuations originating from a tiny volume (~fL). A theoretical model function can be then fitted to the measured auto-correlation curve to obtain physical parameters such as local concentration and diffusion time. However, the application of FCS on membranes is coupled to several difficulties like accurate positioning and stability of the set-up. In this book chapter, we explain the theoretical framework of point FCS and Scanning FCS (SFCS), which is a variation especially suitable for membrane studies. We present a list of materials necessary for SFCS studies on Giant Unilamellar Vesicles (GUVs). Finally, we provide simple protocols for the preparation of GUVs, calibration of the microscope setup, and acquisition and analysis of SFCS data to determine diffusion coefficients and concentrations of fluorescent particles embedded in lipid membranes.

Characterisation of bioaerosol emissions from a Colorado pine forest: results from the BEACHON-RoMBAS experiment

Abstract. The behaviour of primary biological aerosols (PBAs) at an elevated, un-polluted North American forest site was studied using an ultra violet-light induced fluorescence (UV-LIF) measurement technique in conjunction with hierarchical agglomerative cluster analysis (HA-CA). Contemporaneous UV-LIF measurements were made with two wide-band integrated bioaerosol spectrometers, WIBS-3 and WIBS-4, which sampled close to the forest floor and via a continuous vertical profiling system, respectively. Additionally, meteorological parameters were recorded at various heights throughout the forest and used to estimate PBAP (Primary Biological Aerosol Particle) fluxes. HA-CA using data from the two, physically separated WIBS instruments independently yielded very similar cluster solutions. All fluorescent clusters displayed a diurnal minimum at midday at the forest floor with maximum concentration occurring at night. Additionally, the number concentration of each fluorescent cluster was enhanced, to different degrees, during wet periods. A cluster that displayed the greatest enhancement and highest concentration during sustained wet periods appears consistent with behaviour reported for fungal spores. A cluster that appears to be behaviourally consistent with bacteria dominated during dry periods. Fluorescent particle concentrations were found to be greater within the forest canopy than at the forest floor, indicating that the canopy was the main source of these particles rather than the minimal surface vegetation, which appeared to contribute little to overall PBA concentrations at this site. Fluorescent particle concentration was positively correlated with relative humidity (RH), and parameterisations of the aerosol response during dry and wet periods are reported. The aforementioned fungal spore-like cluster displayed a strong positive response to increasing RH. The bacteria-like cluster responded more strongly to direct rain-fall events than other PBA types. Peak concentrations of this cluster are shown to be linearly correlated to the log of peak rainfall rates. Parallel studies by Huffman et al. (2013) and Prenni et al. (2013) showed that the fluorescent particle concentrations correlated linearly with ice nuclei (IN) concentrations at this site during rain events. We discuss this result in conjunction with our cluster analysis to appraise the candidate IN.

Seasonal cycles of fluorescent biological aerosol particles in boreal and semi-arid forests of Finland and Colorado

Abstract. Biological aerosol particles have become increasingly important for atmospheric study, but continuous measurements at high time and size resolution have not been available until recently. Here we report seasonal cycles of fluorescent biological aerosol particles (FBAP) from the boreal forest in Hyytiälä, Finland (18 months) and the semi-arid Manitou Experimental Forest, Colorado (10 months). FBAP at both locations were observed to be highest in summer and lowest in winter, increasing by factors of 12 and 5 between these seasons, respectively. In addition to the low temperatures and reduced sunlight during winter, we suggest that snow cover inhibited FBAP release from local terrestrial surfaces and that more extensive snow cover at the Finland site contributed to lower winter FBAP concentrations. Average size distributions at each site exhibited peaks between 1.5 and 6 μm in aerodynamic diameter. The Finland site consistently showed a dominant, narrow FBAP peak at ~ 3 μm in addition to discreet modes at ~ 1.5 and ~ 5 μm, whereas the Colorado site showed broader peaks at 1.5 and 5 μm, suggesting different modes of biological particles at the two sites. FBAP concentrations in both locations were shown to correlate with daily patterns of relative humidity (RH) during each season. Also during summer at each site, average FBAP concentration scaled with RH, but at the Finland site RH values above ~ 82% led to a significant decrease in FBAP concentration. We hypothesize that this is due to dew formation that inhibits bioparticle release. Lastly we show that rain during summer at each location led to pronounced increases in both fluorescent and total particle concentrations with FBAP peak particle size at ~ 2 μm and concentration scaling with rain intensity. We suggest that these particles are primarily fungal spores and other bioparticles lofted from splashing of rain droplets hitting soil and leaf surfaces. During the summer at the Colorado site we consistently observed a mode of ~ 4 μm particles appearing several hours after rain events that we suggest are fungal spores actively emitted when ambient conditions are most advantageous for spread and germination. The pronounced patterns of fluorescent bioparticles observed here suggest that parameterizations of both daily and seasonal cycles will be important to accurately reflect bioparticle emissions in future studies of atmospheric bioaerosols and their potential effects on clouds and precipitation.

Yearly variation of bacterial production in the Arraial do Cabo protection area (Cabo Frio upwelling region): an evidence of anthropogenic pressure

Arraial do Cabo is where upwelling occurs more intensively on the Brazilian coast. Although it is a protection area it suffers anthropogenic pressure such as harbor activities and sporadic sewage emissions. Short-time studies showed a high variability of bacterial production (BP) in this region but none of them evaluated BP during long periods in a large spatial scale including stations under different natural (upwelling and cold fronts) and anthropogenic pressures. During 2006, we sampled surface waters 10 times (5 in upwelling and 5 in subsidence periods) in 8 stations and we measured BP, temperature as well as the concentrations of inorganic nutrients, pigments and particulate organic matter (POM). BP was up to 400 times higher when sewage emissions were observed visually and it had a positive correlation with ammonia concentrations. Therefore, in 2007, we did two samples (each during upwelling and subsidence periods) during sewage emissions in five stations under different anthropogenic pressure and we also measured particles abundance by flow cytometry. The 12 samples in the most impacted area confirmed that BP was highest when ammonia was higher than 2 μM, also reporting the highest concentrations of chlorophyll a and suspended particles. However, considering all measured variables, upwelling was the main disturbing factor but the pressure of fronts should not be neglected since it had consequences in the auto-heterotrophic coupling, increasing the concentrations of non fluorescent particles and POM. Stations clustered in function of natural and anthropogenic pressures degrees and both determined the temporal-spatial variability.

Biological aerosol particles as a key determinant of ice nuclei populations in a forest ecosystem

AbstractCertain primary biological aerosol particles (PBAPs) are known to have very high ice nucleating ability under mixed‐phase cloud conditions. However, since the abundances of ice nucleation active PBAPs in the atmosphere are generally thought to be extremely small, their importance has remained uncertain. Here we present evidence for the role of PBAPs as atmospheric ice nuclei (IN) active at temperatures ranging from about −34°C to −9°C in a midlatitude ponderosa pine forest ecosystem in summertime. Our measurements show that the number concentrations of IN active at these temperatures were positively correlated with number concentrations of ambient fluorescent biological aerosol particles (FBAPs). Notably, the number concentrations of IN active at warmer temperatures increased quite rapidly in response to increases in the number concentrations of FBAPs. Moreover, we show that a newly‐proposed parameterization related to the number concentrations of FBAPs can better reproduce the number concentrations of IN active over the entire temperature range examined, as compared with parameterizations related solely to the number concentrations of total aerosol particles with diameters larger than 0.5 µm as proposed previously. These results suggest that certain PBAPs released from forest biota can indeed play a key role in determining atmospheric IN populations in this ecosystem, especially at warmer temperatures, potentially leading to ice initiation in nearby mixed‐phase clouds.

Observations of fluorescent and biological aerosol at a high-altitude site in central France

Abstract. Total bacteria, fungal spore and yeast counts were compared with ultraviolet-light-induced fluorescence (UV-LIF) measurements of ambient aerosol at the summit of the Puy de Dôme (PdD) mountain in central France (1465 m a.s.l), which represents a background elevated site. Bacteria, fungal spores and yeast were enumerated by epifluorescence microscopy (EFM) and found to number 2.2 to 23 L−1 and 0.8 to 2 L−1, respectively. Bacteria counts on two successive nights were an order of magnitude larger than in the intervening day. A wide issue bioaerosol spectrometer, version 3 (WIBS-3) was used to perform UV-LIF measurements on ambient aerosol sized 0.8 to 20 μm. Mean total number concentration was 270 L−1 (σ = 66 L−1), found predominantly in a size mode at 2 μm for most of the campaign. Total concentration (fluorescent + non-fluorescent aerosol) peaked at 500 L−1 with a size mode at 1 μm because of a change in air mass origin lasting around 48 h. The WIBS-3 features two excitation and fluorescence detection wavelengths corresponding to different biological molecules, although non-biological interferents also contribute. The mean fluorescent particle concentration after short-wave (280 nm; associated with tryptophan) excitation was 12 L−1 (σ = 6 L−1), and did not vary much throughout the campaign. In contrast, the mean concentration of particles fluorescent after long-wave (370 nm; associated with NADH) excitation was 95 L−1 (σ = 25 L−1), and a nightly rise and subsequent fall of up to 100 L−1 formed a strong diurnal cycle in the latter. The two fluorescent populations exhibited size modes at 3 μm and 2 to 3 μm, respectively. A hierarchical agglomerative cluster analysis algorithm was applied to the data and used to extract different particle factors. A cluster concentration time series representative of bacteria was identified. This was found to exhibit a diurnal cycle with a maximum peak appearing during the day. Analysis of organic mass spectra recorded using an aerosol mass spectrometer (AMS; Aerodyne Inc.) suggests that aerosol reaching the site at night was more aged than that during the day, indicative of sampling the residual layer at night. Supplementary meteorological data and previous work also show that PdD lies in the residual layer/free troposphere at night, and this is thought to cause the observed diurnal cycles in organic-type and fluorescent aerosol particles. Based on the observed disparity between bacteria and fluorescent particle concentrations, fluorescent non-PBA is likely to be important in the WIBS-3 data and the surprisingly high fluorescent concentration in the residual layer/free troposphere raises questions about a ubiquitous background in continental air during the summer.

Fluorescent biological aerosol particles measured with the Waveband Integrated Bioaerosol Sensor WIBS-4: laboratory tests combined with a one year field study

Abstract. In this paper bioaerosol measurements conducted with the Waveband Integrated Bioaerosol Sensor mark 4 (WIBS-4) are presented. The measurements comprise aerosol chamber characterization experiments and a one-year ambient measurement period at a semi-rural site in South Western Germany. This study aims to investigate the sensitivity of WIBS-4 to biological and non-biological aerosols and detection of biological particles in the ambient aerosol. Several types of biological and non-biological aerosol samples, including fungal spores, bacteria, mineral dust, ammonium sulphate, combustion soot, and fluorescent polystyrene spheres, were analyzed by WIBS-4 in the laboratory. The results confirm the sensitivity of the ultraviolet light-induced fluorescence (UV-LIF) method to biological fluorophores and show the good discrimination capabilities of the two excitation wavelengths/detection wavebands method applied in WIBS-4. However, a weak cross-sensitivity to non-biological fluorescent interferers remains and is discussed in this paper. All the laboratory studies have been undertaken in order to prepare WIBS-4 for ambient aerosol measurements. According to the one-year ambient aerosol study, number concentration of fluorescent biological aerosol particles (FBAP) show strong seasonal and diurnal variability. The highest number concentration of FBAP was measured during the summer term and decreased towards the winter period when colder and drier conditions prevail. Diurnal FBAP concentrations start to increase after sunset and reach maximum values during the late night and early morning hours. On the other hand, the total aerosol number concentration was almost always higher during daytime than during nighttime and a sharp decrease after sunset was observed. There was no correlation observed between the FBAP concentration and the meteorological parameters temperature, precipitation, wind direction and wind speed. However, a clear correlation was identified between the FBAP number concentration and the relative humidity. Humidity-controlled release mechanisms of some fungal spore species are discussed as a possible explanation.

Millimeter scale profiles of chlorophyll fluorescence: Deciphering the microscale spatial structure of phytoplankton

Marine food webs and biogeochemical cycles are driven by interactions between individual phytoplankton and other micro-organisms embedded within turbulent flows. Understanding the causes and ecological consequences of these interactions requires measurement of the spatial distribution of organisms across sub-meter scales relevant to their activities. However, estimates of many microscale processes (e.g., encounter rates, competition) are implicitly based on a random distribution of plankton despite increasing evidence of patchy distributions of turbulence and phytoplankton at the oceans microscale. Further complicating our understanding of microscale phytoplankton ecology, recent studies have suggested that the high levels of fluorescence variability measured at sub-centimeter scales may be due to the detection of separate, large phytoplankton particles (i.e. large cells, chains and aggregates) rather than ‘patches’ of increased cell abundances. By comparing coincident fluorescence estimates measured with millimeter (μL) and centimeter (mL) scale resolution, we show that estimates of phytoplankton biomass made at centimeter scales are consistent with averaging discrete variations in fluorescence measured at millimeter scales and that a critical scale exists where measures of fluorescence variability transitions from representing an individual to a patch. Application of nearest neighbor analysis to the discrete fluorescence patterns showed deviations from complete spatial randomness towards clustering across scales of millimeters to tens of centimeters. The strength of the deviation from random increased significantly in regions of elevated phytoplankton concentrations. No relationship was observed between fluorescent particle concentrations or nearest neighbor distances with the rate of dissipation of turbulent kinetic energy. Our results provide empirical evidence that the scale at which phytoplankton distributions are estimated by chlorophyll fluorescence may be critical to the interpretation of microscale phytoplankton spatial patterns and that biological processes may strongly influence the microscale structure and ecology of marine systems.

An Elegant Way to Quantitatively Analyze Oligomer Formation in Solution

An Elegant Way to Quantitatively Analyze Oligomer Formation in Solution

The fluorescence properties of aerosol larger than 0.8 μm in urban and tropical rainforest locations

Abstract. UV-LIF measurements were performed on ambient aerosol in Manchester, UK (urban city centre, winter) and Borneo, Malaysia (remote, tropical) using a Wide Issue Bioaerosol Spectrometer, version 3 (WIBS3). These sites are taken to represent environments with minor and significant primary biological aerosol (PBA) influences respectively, and the urban dataset describes the fluorescent background aerosol against which PBA must be identified by researchers using LIF. The ensemble aerosol at both sites was characterised over 2–3 weeks by measuring the fluorescence intensity and optical equivalent diameter (DP) of single particles sized 0.8 ≤ DP ≤ 20 μm. Filter samples were also collected for a subset of the Manchester campaign and analysed using energy dispersive X-Ray (EDX) spectroscopy and environmental scanning electron microscopy (ESEM), which revealed mostly non-PBA at D ≤ 1 μm. The WIBS3 features three fluorescence channels: the emission following a 280 nm excitation is recorded at 310–400 nm (channel F1) and 400–600 nm (F2), and fluorescence excited at 350 nm is detected at 400–600 nm (F3). In Manchester the primary size mode of fluorescent and non-fluorescent material was present at 0.8–1.2 μm, with a secondary fluorescent mode at 2–4 μm. In Borneo non-fluorescent material peaked at 0.8–1.2 μm and fluorescent at 3–4 μm. Agreement between fluorescent number concentrations in each channel differed at the two sites, with F1 and F3 reporting similar concentrations in Borneo but F3 outnumbering F1 by a factor of 2–3 across the size spectrum in Manchester. The fluorescence intensity in each channel generally rose with DP at both sites with the exception of F1 intensity in Manchester, which peaked at DP = 4 μm, causing a divergence between F1 and F3 intensity at larger DP. This divergence and the differing fluorescent particle concentrations demonstrate the additional discrimination provided by the F1 channel in Manchester. The relationships between fluorescence intensities in different pairs of channels were also investigated as a function of DP. Differences between these metrics were apparent at each site and provide some distinction between the two datasets. Finally, particle selection criteria based on the Borneo dataset were applied to identify a median concentration of 10 "Borneo-like" fluorescent particles per litre in Manchester.

Analysis of the HTLV-1 Gag assembly pathway by biophysical fluorescence

Much of the mechanistic details for how HTLV-1 Gag orchestrates virus particle assembly and release are poorly understood. Here, we monitored the behavior of both membrane-bound and cytoplasmic HTLV-1 Gag in real-time in living cells incubated on a fluorescence microscope. We used both fluorescence fluctuation spectroscopy (FFS, conventional and z-scan) and fluorescence imaging (epi-illumination, total internal reflection fluorescence (TIRF)) to investigate the relationship between cytoplasmic and membrane bound Gag, using a Gag-YFP model system. FFS determines the brightness, mobility, and concentration (conventional) and localization (z-scan) of fluorescent particles from the intensity bursts generated by individual particles passing through a small observation volume, which yields information about protein stoichiometry, interactions, transport, and distribution. By coupling the single-molecule FFS technique with imaging techniques capable of monitoring Gag localization (epi-illumination) and membrane-specific localization (TIRF), we achieved new insights into the earliest events in HTLV-1 Gag assembly, and differences to HIV-1 Gag. We found that HTLV-1 Gag membrane-targeting occurred at all cytoplasmic concentrations measured, while appreciable membrane-targeting for HIV-1 required Gag cytoplasmic concentration to exceed a threshold. In addition, z-scan FFS revealed that a substantial population of membrane-bound HTLV-1 Gag exists not as puncta, but as a diffuse, low-order, dynamic “sheet.” These observations, coupled with previous observations of cytoplasmic Gag interactions and mobility, point to differences in membrane targeting of HTLV-1 and HIV-1 Gag. In summary, the suite of biophysical fluorescence techniques, applied HTLV-1 Gag, provide unparalleled information concerning HTLV-1 Gag trafficking processes in vivo, elucidating assembly pathway differences between HTLV-1 and other retroviruses.

Cryptic peaks: invisible vertical structure in fluorescent particles revealed using a planar laser imaging fluorometer

Small‐scale vertical structure in the distribution of phytoplankton could be fundamentally important for zooplankton foraging, trophic coupling, and carbon cycling in planktonic ecosystems. Here, we identify regions of structure in phytoplankton communities that would be undetected by fluorometers by comparing the vertical distribution of chlorophyll fluorescence to the concentration of fluorescent particles over submeter scales. Images acquired with a free‐falling planar laser fluorescence imaging system were used to calculate vertical profiles of the concentrations and spatial distributions of fluorescent particles (e.g., eukaryotic phytoplankton, aggregates) and bulk fluorescence. We frequently observed peaks in the concentration of fluorescent particles with no coincident peak in bulk fluorescence: we define these features as cryptic peaks. These cryptic peaks can occur because the integrated fluorescence of the particles that are resolved by the imaging system is a small fraction of the total fluorescence; thus, a dramatic local change in the abundance of fluorescent particles can occur without significantly changing the bulk fluorescence. We also observed bulk fluorescence‐only peaks: peaks in bulk fluorescence with no coincident peak in fluorescent particle concentration. These features suggest that peaks in bulk fluorescence or chlorophyll a do not necessarily indicate increases in the concentration of the fluorescent particles resolved by our system, again emphasizing the difference between these two measures of phytoplankton structure. Comparing the relative abundances of two size classes of the fluorescent particles in the images revealed that the size composition of the fluorescent particles also varied over small scales. Phytoplankton less than ~ 500 µm in length numerically dominated the composition of most (65%) of the cryptic peaks we observed. By comparing vertical profiles of fluorescent particle concentration from two drops separated by less than an hour, we hypothesize that the peaks formed through vertical shearing of existing patches. Cryptic peaks contained almost 20% of the total number of fluorescent particles counted in all drops during our study and thus could represent disproportionately intense regions for important ecological processes relative to the rest of the water column.

Predicting Protein Co-Expression Fractions in Living Cells

Fluorescence fluctuation spectroscopy utilizes the fluctuation in a fluorescent signal to determine molecular brightness, concentration, and diffusion properties of fluorescent particles passing through an optical volume. Brightness analysis is used to quantify the stoichiometry of protein association and to determine the number of proteins in a complex. In protein hetero-dimerization studies, it is highly desirable to be able to control protein co-expression fractions. The expression of each protein should be selected in order to maximize the likelihood of protein association. Yet protein expression is the output factor from a lipid-mediated transfection process in which only the plasmid DNA (input parameter) can be explicitly controlled. In this work we develop and test a working model which relates the protein expression fraction of two proteins to the plasmid DNA mixing fraction. Experimentally, we express two differently colored fluorescent proteins in CV-1 cells and split their emitted signal into two different detection channels based on color. The intensity fraction from the two channels is used to determine the relative amount of expressed protein in the cell. The experiments show that the DNA mixing fraction determines the average protein expression fraction. We extend this work by examining the cell-to-cell variation in protein expression and constructing a simple model which relates the expression variation to the average number of active plasmids. This study provides insight into the fundamentals of lipid-mediated transfection and demonstrates that we can control protein co-expression fractions through selection of plasmid DNA mixing fractions. This work is supported by NIH grant R01GM064589.

Protein-mRNA Interactions Observed in Living Cells By Dual-Color Fluorescence Fluctuation Spectroscopy

RNA binding protein controls the localization, translation and degradation of mRNA of specific proteins, therefore exerting a key influence on the cell metabolism, motility and differentiation. The zipcode binding protein 1 (ZBP1) binds to the 3′ untranslated region (UTR) of beta-actin mRNA and regulates its translation. Quantitative characterization of the interaction between mRNA and protein is crucial to dissect this regulation mechanism. We have developed a dual-color fluorescence fluctuation spectroscopy (FFS) technique to study the interaction between mRNA and protein directly in living cells. FFS determines the brightness, concentration and diffusion time of fluorescent particles from the intensity bursts generated by individual particles passing through a small observation volume. Dual-color FFS distinguishes fluorescent species by brightness and diffusion time as well as the fluorescence color. In this study, we apply dual-color FFS to study the interaction between beta-actin mRNA and ZBP1. The endogenous mouse beta-actin mRNA is visualized by incorporating 24 MS2 stem-loops in the 3′ UTR, which are specifically bound by fluorescently labeled MS2 coat protein (MCP). We have developed a tandem dimeric MCP system that is particularly suitable for FFS brightness analysis. Since multiple MCPs bind to a single mRNA, the brightness of mRNA is significantly higher than that of free MCP, which allows us to readily resolve mRNA from free MCPs. Furthermore, ZBP1 is labeled with a different color and dual-color FFS directly extracts the interacting mRNA-ZBP1 species. The interaction is visualized by brightness signatures and the binding affinity is extracted by measuring different cells with varying concentrations of ZBP1. This work is supported by National Institute of Health EB2060.

Safranine O as a fluorescent probe for mitochondrial membrane potential studied on the single particle level and in suspension

The permeant cationic dye safranine O is often used to measure mitochondrial membrane potential due to the dependence of both its absorption and fluorescence on mitochondrial energization, which causes its oligomerization inside mitochondria. In the present study we have used fluorescent correlation spectroscopy (FCS) to record the fluorescence changes on a micro level, i.e. under conditions permitting resolution of contributions from single particles (molecules of the dye and stained mitochondria). We have shown that the decrease in fluorescence signal from a suspension of energized mitochondria stained with a high safranine concentration (10 microM) is explained by the decrease in dye concentration in the medium in parallel with the accumulation of the dye inside the mitochondria, which results in fluorescence quenching. With 1 microM safranine O, the fluorescence rise after energization is caused by the accumulation of the dye up to a level not sufficient for full fluorescence quenching and also by the higher intensity of mitochondrial fluorescence on immersion of the dye in the hydrophobic milieu. Besides the estimation of the inner mitochondrial membrane potential, this approach also assesses the concentration of fluorescent particles. The non-monotonic dependence of the FCS parameter 1/G(tau-->0) on the concentration of mitochondrial protein suggests heterogeneity of the system with respect to fluorescence of particles. An important advantage of the described method is its high sensitivity, which allows measurements with low concentrations and quantities of mitochondrial protein in samples (less than 10 microg).

Fluorescence Fluctuation Spectroscopy on Viral-Like Particles Reveals Variable Gag Stoichiometry

Fluorescence fluctuation spectroscopy determines the brightness, size, and concentration of fluorescent particles from the intensity bursts generated by individual particles passing through a small observation volume. Brightness provides a measure of the number of fluorescently labeled proteins within a complex and has been used previously to determine the stoichiometry of small oligomers in cells. We extend brightness analysis to large macromolecular protein complexes containing thousands of proteins and determine their stoichiometry. This study investigates viral-like particles (VLP) formed from human immunodeficiency virus type 1 (HIV-1) Gag protein expressed in COS-1 cells using fluorescence fluctuation spectroscopy to determine the stoichiometry of HIV-1 Gag within the particles. Control experiments establish that the stoichiometry and size of VLPs are not influenced by labeling of HIV-1 Gag with a fluorescent protein. The experiments further show that the brightness scales linearly with the amount of labeled Gag within the particle. Brightness analysis shows that the Gag stoichiometry of VLPs formed in COS-1 cells is not constant, but varies with the amount of transfected DNA plasmid. We observed HIV-1 Gag stoichiometries ranging from ∼750 to ∼2500, whereas the size of the VLPs remains unchanged. This result indicates that large areas of the VLP membrane are void of Gag protein. Therefore, a closed layer of HIV-1 Gag at the membrane is not required for VLP production. This study shows that brightness analysis has the potential to become an important tool for investigating large molecular complexes by providing quantitative information about their size and composition.

Spectrally resolved fluorescence correlation spectroscopy based on global analysis.

Multicolor fluorescence correlation spectroscopy has been recently developed to study chemical interactions of multiple chemical species labeled with spectrally distinct fluorophores. In the presence of spectral overlap, there exists a lower detectability limit for reaction products with multicolor fluorophores. In addition, the ability to separate bound product from reactants allows thermodynamic properties such as dissociation constants to be measured for chemical reactions. In this report, we utilize a spectrally resolved two-photon microscope with single-photon counting sensitivity to acquire spectral and temporal information from multiple chemical species. Further, we have developed a global fitting analysis algorithm that simultaneously analyzes all distinct auto- and cross-correlation functions from 15 independent spectral channels. We have demonstrated that the global analysis approach allows the concentration and diffusion coefficients of fluorescent particles to be resolved despite the presence of overlapping emission spectra.

Performance of UVAPS with respect to detection of airborne fungi

This paper studies the detection limit, selectivity and counting efficiency of an ultraviolet aerodynamic particle sizer spectrometer (UVAPS) with regard to aerosolized fungal spores. The study demonstrated the ability of the instrument for detection and measurement of fungal spores under controlled conditions. A reasonable correlation was found between the UVAPS and the AGI-30 impinger in measuring the aerosol fungal spore concentrations under investigation: Penicillium and Aspergillus niger ( r = 0.911 , p < 0.005 and r = 0.882 , p < 0.05 , respectively). A linear relationship between total particle concentration and fluorescent particle concentration was found in the range from 0 to 70 particles / cm 3 . Its lower detection limit was found to be 0.01 particles / cm 3 . The dry generation method which was used for generating fungal spores has proved to be reproducible and easy to control, as well as simple and inexpensive.