High throughput particle analysis: combining dielectrophoretic particle focussing with confocal optical detection

A micro flow cytometer has been fabricated that detects and counts fluorescent particles flowing through a microchannel at high speed based upon their fluorescence emission intensity. Dielectrophoresis is used to continuously focus particles within the flowing fluid stream into the centre of the device, which is 40 microm high and 250 microm wide. The method ensures that all the particles pass through an interrogation region approximately 5 microm in diameter, which is created by focusing a beam of light into a spot. The functioning of the device was demonstrated by detecting and counting fluorescent latex particles at a rate of up to 250 particles/s. A mixture of three different populations of latex particle was used, each sub-population with a distinct level of fluorescent intensity. The device was evaluated by comparison with a conventional fluorescent activated cell sorter (FACS) and numerical simulation demonstrated that for 6 microm beads, and for this design of chip the theoretical throughput is of the order of 1000 particles/s (corresponding to a particle velocity of 10 mm s(-1)).     

Solubilization of active green fluorescent protein from insoluble particles by guanidine and arginine

Expression of green fluorescent protein (GFP) in Escherichia coli (E. coli) resulted in only small amount of soluble and fluorescent GFP protein and hence most of the protein in insoluble particles. The expressed GFP in insoluble particles, however, was fluorescent, indicating that it is at least in part folded with an intact chromophore. The GFP in insoluble particles could not be solubilized by an aqueous (denaturant-free) buffer. Solubilization of active GFP from insoluble particles was then attempted with guanidine hydrochloride (GdnHCl), a strong protein-denaturant, or L-arginine, an aggregation suppressor. Solubilization from insoluble particles by 6M GdnHCl led to complete denaturation of the GFP existing in insoluble particles, while GdnHCl solution at lower concentration could solubilize fluorescent GFP. Solubilization of fluorescently active GFP from insoluble particles was also achieved by L-arginine. It is noteworthy that L-arginine was stronger in solubilizing insoluble GFP than GdnHCl below 2M. These results demonstrate that some proteins expressed in E. coli may form insoluble particles containing native conformation and L-arginine may be used to recover the proteins in the native form from such insoluble particles.     

Flow cytometric measurement of rates of particle uptake from dilute suspensions by a ciliated protozoan

Flow cytometry is used to measure rates of ingestion of particles from dilute monodisperse suspensions by the ciliate Tetrahymena pyriformis. The particles used are polystyrene microspheres containing a fluorescent dye. Measurements were made directly, that is, by determining the fluorescence intensities from microspheres ingested by cells in samples collected from the experimental feeding apparatus. The fact that fluorescence intensities from individual cells can be grouped into discrete classes based on the numbers of fluorescent particles associated with the cells makes it possible to calibrate the flow cytometer and convert fluorescence measurements into numbers of particles ingested by average cells. At low particle concentration or high ciliate concentration, ingestion data must be corrected for depletion of particles during the assay, and a method for doing this is described. Experiments at various ciliate concentrations show that ingestion rates are not affected by this concentration. The methods developed should allow measurements of rates of ingestion of particles from concentrated and polydisperse suspensions. For such measurements, nonfluorescent particles together with a fraction of fluorescent tracer particles would be used.     

High expression of green fluorescent protein in Pichia pastoris leads to formation of fluorescent particles

Wild type gene for green fluorescent protein (GFP) was stably integrated into the Pichia pastoris genome and yielded an expression level of over 40% of total cellular protein. The high cytoplasmic concentration of fluorescent (properly folded and processed) GFP caused the formation of fluorescent spherical structures, which could be observed by fluorescence or confocal microscopy after controlled permeabilization of the yeast cells with 0.2% N-lauroyl sarcosine (NLS). Fluorescent GFP particles were also isolated after removal of the cell wall and found to be quite resistant to 0.2% N-lauroyl sarcosine. SDS-PAGE analysis of the isolated fluorescent particles revealed the presence of an 80 kDa protein (alcohol oxidase) and GFP (30%). We conclude that GFP is able to enter spontaneously into the peroxisomes and is inserted into densely packed layers of alcohol oxidase. Consequently, the formation of similar fluorescent particles can also be expected in other organisms when using high-level expression systems. As GFP is widely used in fusion with other proteins as a reporter for protein localization and for many other applications in biotechnology, care must be taken to avoid false interpretations of targeting or trafficking mechanisms inside the cells. In addition, when whole cells or cytoplasmic fractions are used for the quantitative determination of GFP levels, incorrect and misleading values of GFP could be obtained due to the formation of fluorescent particles containing material inside which is not available for fluorescence measurements.     

Preparation of fluorescently labelled hybrid histone octamers

Labelling hybrid histone octamers (the Cys variant of histone H4 replaced histone H4 in the chicken erythrocyte octamer) with the fluorescent probe 5-(2(iodoacetyl)aminoethyl)aminonapthalene- 1-sulfonic acid, IAEDANS, resulted in significant non-specific incorporation of label. Fluorescently labelled hybrid histone octamers were prepared by reconstitution methodology after labelling the isolated histone Cys-H4 and separation of specifically and non-specifically labelled histone. Core particles prepared from these octamers have identical thermal denaturation to unlabelled core particles demonstrating that the incorporation of a fluorescent probe at this site has no overall effect on either histone-histone or histone-DNA interactions. DNase 1 digestion of 32P end-labelled fluorescent core particles yielded the anticipated asymmetric cutting pattern with a 10 bp interval between fragments. Comparison of the cutting pattern with those previously obtained in these laboratories for both polyglutamic acid reconstituted and 'native' core particles demonstrated that fluorescent core particles had an enhanced susceptibility to digestion at site 7.     

Localization of fluorescent analogs of cholesterol and its esters in lipid models of membranes and lipoproteins

The localization of the fluorescent cholesterol analogue--delta 5,7,9(11)-cholestatrien-3 beta-ol (B-CTE) and its methyl (M-CTE) and stearoyl (St-CTE) esters in model lipid particles were investigated by the radiationless energy transfer. It is shown that 67-100% of B-CTE molecules localize in the phospholipid monolayer of particles containing phosphatidylcholine and triolein (1:2 w/w). The replacement of a hydrogen atom in the hydroxyl by methyl resulted in immersion of 2/3 of the M-CTE molecules to the triolein core. This fact confirmed distribution of the fluorescent probe molecules in the whole volume of lipid particles. St-CTE was practically completely localized in the core of lipid particles. The results obtained evidence for the important role of 3-OH group in keeping the B-CTE molecules in the phospholipid monolayer of the investigated particles.     

Supramolecular organization of heteroxylan-dehydrogenation polymers (synthetic lignin) nanoparticles

The supramolecular organization of particles composed of heteroxylans (HX) and synthetic lignin (dehydrogenation polymer, DHPs) was studied by light scattering (LS), atomic force microscopy (AFM), and fluorescent probes. Results from static and quasi-elastic light scattering indicate a dense core surrounded by a soft corona. Such organization is also supported by AFM images of the particles that display Gaussian height profiles when a low tapping force is applied, whereas the shape of the profile obtained at a higher mechanical solicitation is irregular and sharp due to deformation of the particles resulting from the tip indentation. This suggests a difference in mechanical behavior between the inner and outer parts of the particles. The formation of local chemical heterogeneities was demonstrated by use of two fluorescent polarity probes (pyrene and methyl-amino-pyrene) to be induced by the core-corona organization.     

A fluorescent derivative of ribosomal protein S18 which permits direct observation of messenger RNA binding.

70 S Escherichia coli ribosomes were reacted with the fluorescent dye N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid for 10 min under mild conditions. The resulting ribosomes were fully active. 30 S subunits isolated from these particles were also fully active. They contain approximately 0.7 eq of fluorescent dye. Nearly all of it is attached to protein S18. Competitive reaction with N-ethylmaleimide implies that the fluorescent dye is located at cysteine 10 of the protein. The labeled 30 S particles will recombine with 50 S subunits to form stable 70 S particles. Thus the procedures we have developed allow the large scale preparation of an active fluorescent conjugate of the 70 S ribosome. The fluorescence of the 70 S particles is sensitive to the binding of mRNA, showing both quenching and a shift in emission spectra. Thus it affords a simple way to quantitate mRNA binding directly. In pilot studies without tRNA, the binding constant of the initiation triplet codon adenylyl-(3' leads to 5')-uridylyl-(3' leads to 5')-guanosine to 70 S ribosome was found to be an order of magnitude larger than that of polyuridylic acid.     

Fluorescent silica nanospheres for digital counting bioassay of the breast cancer marker HER2/neu [correction of HER2/nue

This paper describes the use of fluorescent silica nanospheres as luminescent signal amplifiers in biological assays based on digital counting of individual particles instead of measuring averaged fluorescence intensity. We recently described a simple method to prepare highly fluorescent mono-dispersed silica nanospheres that avoids microemulsion formulations and the use of surfactants. Modification of the St?ber method was used successfully to prepare fluorescent silica spheres with the inorganic dye dichlorotris(1,10-phenanathroline)ruthenium (II) hydrate encapsulated during the condensation of tetraethylorthosilicate in ethanol and dye aqueous mixtures. Modifications in the ammonia and water content in the reaction mixture resulted in mono-dispersed silica spheres of 65, 440 and 800 nm in diameter. The dye-encapsulating particles emit intense red luminescence when excited at 460 nm. We observed an increased photostability and longer fluorescence lifetime in our particles that we attributed to increased protection of the encapsulated dye molecules from molecular oxygen. The newly prepared fluorescent silica particles were easily modified using trialkoxysilane reagents for covalent conjugation of anti-HER2/neu. We demonstrated the utility of the fluorescent nanospheres to detect the cancer marker HER2/neu in a glass slide based assay. The assay was shown to be simple but highly sensitive with a limit of detection approaching 1 ng/mL and a linear range between 1 ng/mL and 10 microg/mL of HER2/neu.     

Polymerizable fluorescein derivatives: synthesis of fluorescent particles and their cellular uptake

Fluorescent particles are used for a diverse number of biochemical assays including intracellular imaging, cellular tracking, as well as detection of a variety of biomolecules. They are typically prepared by postpolymerization conjugations of dyes onto preformed particles. Herein we report the synthesis of aminomethyl-functionalized fluorescent particles via the synthesis and application of polymerizable fluorescein monomers. These monomers allowed high and controllable fluorophore loading into the particles, resulting in enhanced fluorescence properties in comparison with more commonly used carboxyfluorescein conjugated particles. Furthermore, the particles were rapidly taken up by cells with enhanced fluorescence. The herein presented results demonstrate the advantages of dye polymerization in contrast with more conventional conjugation strategies for fluorescent particle generation with applications in the life sciences.     

Multifunctional protein particles with dual analytical channels for colorimetric enzymatic bioassays and fluorescent immunoassays

Advanced multifunctional protein particles encapsulated enzymes and antibodies were developed for enzymatic bioassays and immunoassays with colorimetric and fluorescent channels. A colorimetric channel based on color-substrate precipitation was assigned for enzymatic bioassays for the measurement of hydrogen peroxide with the lowest detectable concentration of 10 μM. A fluorescent channel based on fluorescent labeled antibodies was assigned for immunoassays for the measurement of mouse immunoglobulin G (M IgG) with the lowest detectable concentration of 1.25 μgL(-1). The protein microparticles were fabricated with a template-assisted self-assembly technique termed "Protein Activation Spontaneous Self-assemble" (PASS). The multifunctional protein particles prepared with the PASS method have the advantages of high loading of analytical biomolecules, integrated biological functions, porous structure, and more importantly, they are optically transparent and fluorescence inactive. These unique features make our protein particles a new generation of bead-based platforms to perform enzyme bioassays and immunoassays.     

A test of granulocyte membrane integrity and phagocytic function.

An assay of granulocyte viability has been developed which yields information about rwo important cell parameters, cell membrane integrity and phagocytic activity. The assay is based on the fact that only live cells can accumulate fluorescein, which is enzymatically split from the nonfluorescent substrate fluorescein diacetate. Dead cells, on the other hand, become permeable to the fluorescent red dye ethidium bromide. When cells are exposed first to opsonized zymosan particles, which they can phagocytize, then to a combination of these fluorescent dyes, one can distinguish microscopically between dead cells with fluorescent red nuclei, live cells which fluoresce green, and live cells with phagocytic function which are swollen with the pink zymosan particles in a green fluorescing cytoplasm. This assay takes 20--30 min and can be used to distinguish different degrees of cellular damage after cryopreservation.     

A novel approach to nonradioactive hybridization assay of nucleic acids using stained latex particles.

The paper describes a sensitive latex hybridization assay (LHA) method applied for indirect detection of biotinylated nucleic acid hybrids immobilized on a synthetic membrane. The biotinylated hybrids were visualized by means of latex particles containing the fluorescent dye pyronine G and coated with streptavidin; 1.6 and 0.3 pg of lambda-phage DNA was detected by dot blot hybridizations on nylon membrane and polyethyleneimine-cellophane, respectively. The assay sensitivity was increased by three orders of magnitude over that with fluorescently labeled probes due to encapsulation of the fluorescent dye in polymer particles. LHA is simple (single-stage detection procedure), fast, and more sensitive than any of the other nonradioactive hybridization methods.     

Disassembly of structurally modified viral nanoparticles: characterization by fluorescence correlation spectroscopy

Analysis of the breakdown products of engineered viral particles can give useful information on the particle structure. We used various methods to breakdown both a recombinant enveloped virus and virus-like particles (VLPs) from two non-enveloped viruses and analysed the resulting subunits by fluorescence correlation spectroscopy (FCS). Analysis of the enveloped baculovirus, Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), displaying the green fluorescent protein (GFP) fused to its envelope protein gp64 was performed in the presence and absence of 5 mM SDS and 25 mM DTT. Without treatment, the viral particle showed a diffusion time of 3.3 ms. In the presence of SDS, fluorescent subunits with diffusion times of 0.2 ms were observed. Additional treatment with DTT caused a drop in the diffusion time to 0.1 ms. Changes in the amplitude of the autocorrelation function suggested a 3-fold increase in fluorescent particle number when viral particles were treated with SDS, and a further 1.5-fold increase with additional treatment with DTT. Thus, the data showed that an average of 4.5 molecules of gp64-GFP was incorporated in the membrane of the modified baculovirus. Further, this suggests that each fluorescent gp64 trimer carries on average 1.5 fluorescent units. Similar experiments were carried out with two non-enveloped fluorescent virus-like particles (fVLPs) that displayed enhanced green fluorescent protein (EGFP). These, fVLPs of canine and human B19 parvoviruses were treated with 6 M urea and 5 mM SDS, respectively. Correspondingly, the original hydrodynamic radii of 17 and 14 nm were reduced to 9 and 5 nm after treatment. Here, the change in the amplitude of the autocorrelation curve suggested a 10-fold increase in particle number when viral particles of CPV were treated with 6 M urea at 50 degrees C for 10 min. For EGFP-B19, there was a decrease in the amplitude, accompanied by a 9-fold increase in the number of fluorescent units with SDS treatment. The results showed that approximately 10 and 9 fluorescent units were associated with the corresponding CPV and B19 VLPs. In summary, we were able to estimate the number of fluorescent subunits in a baculovirus containing a GFP-fusion with its gp64 envelope protein and in two different parvo-VLPs containing EGFP-fused with their VP2 capsid proteins.     

Individual rotavirus-like particles containing 120 molecules of fluorescent protein are visible in living cells

Rotaviruses are large, complex icosahedral particles consisting of three concentric capsid layers. When the innermost capsid protein VP2 is expressed in the baculovirus-insect cell system it assembles as core-like particles. The amino terminus region of VP2 is dispensable for assembly of virus-like particles (VLP). Coexpression of VP2 and VP6 produces double layered VLP. We hypothesized that the amino end of VP2 could be extended without altering the auto assembly properties of VP2. Using the green fluorescent protein (GFP) or the DsRed protein as model inserts we have shown that the chimeric protein GFP (or DsRed)-VP2 auto assembles perfectly well and forms fluorescent VLP (GFP-VLP2/6 or DsRed-VLP2/6) when coexpressed with VP6. The presence of GFP inside the core does not prevent the assembly of the outer capsid layer proteins VP7 and VP4 to give VLP2/6/7/4. Cryo-electron microscopy of purified GFP-VLP2/6 showed that GFP molecules are located at the 5-fold vertices of the core. It is possible to visualize a single fluorescent VLP in living cells by confocal fluorescent microscopy. In vitro VLP2/6 did not enter into permissive cells or in dendritic cells. In contrast, fluorescent VLP2/6/7/4 entered the cells and then the fluorescence signal disappear rapidly. Presented data indicate that fluorescent VLP are interesting tools to follow in real time the entry process of rotavirus and that chimeric VLP could be envisaged as "nanoboxes" carrying macromolecules to living cells.     

Nanoparticles for the delivery of genes and drugs to human hepatocytes

Hepatitis B virus envelope L particles form hollow nanoparticles displaying a peptide that is indispensable for liver-specific infection by hepatitis B virus in humans. Here we demonstrate the use of L particles for the efficient and specific transfer of a gene or drug into human hepatocytes both in culture and in a mouse xenograft model. In this model, intravenous injection of L particles carrying the gene for green fluorescent protein (GFP) or a fluorescent dye resulted in observable fluorescence only in human hepatocellular carcinomas but not in other human carcinomas or in mouse tissues. When the gene encoding human clotting factor IX was transferred into the xenograft model using L particles, factor IX was produced at levels relevant to the treatment of hemophilia B. The yeast-derived L particle is free of viral genomes, highly specific to human liver cells and able to accommodate drugs as well as genes. These advantages should facilitate targeted delivery of genes and drugs to the human liver.     

Human cytomegalovirus labeled with green fluorescent protein for live analysis of intracellular particle movements

Human cytomegalovirus (HCMV) replicates in the nuclei of infected cells. Successful replication therefore depends on particle movements between the cell cortex and nucleus during entry and egress. To visualize HCMV particles in living cells, we have generated a recombinant HCMV expressing enhanced green fluorescent protein (EGFP) fused to the C terminus of the capsid-associated tegument protein pUL32 (pp150). The resulting UL32-EGFP-HCMV was analyzed by immunofluorescence, electron microscopy, immunoblotting, confocal microscopy, and time-lapse microscopy to evaluate the growth properties of this virus and the dynamics of particle movements. UL32-EGFP-HCMV replicated similarly to wild-type virus in fibroblast cultures. Green fluorescent virus particles were released from infected cells. The fluorescence stayed associated with particles during viral entry, and fluorescent progeny particles appeared in the nucleus at 44 h after infection. Surprisingly, strict colocalization of pUL32 and the major capsid protein pUL86 within nuclear inclusions indicated that incorporation of pUL32 into nascent HCMV particles occurred simultaneously with or immediately after assembly of the capsid. A slow transport of nuclear particles towards the nuclear margin was demonstrated. Within the cytoplasm, most particles performed irregular short-distance movements, while a smaller fraction of particles performed centripetal and centrifugal long-distance movements. Although numerous particles accumulated in the cytoplasm, release of particles from infected cells was a rare event, consistent with a release rate of about 1 infectious unit per h per cell in HCMV-infected fibroblasts as calculated from single-step growth curves. UL32-EGFP-HCMV will be useful for further investigations into the entry, maturation, and release of this virus.     

Competitive cellular uptake of nanoparticles made from polystyrene, poly(methyl methacrylate), and polylactide

The uptake behavior of negatively charged fluorescent nanoparticles made from different polymers (PS, PMMA, and PLLA) is studied on HeLa cells. All particles are obtained by the miniemulsion process using sodium dodecylsulfate as anionic surfactant. The size of the particles is in the range 105-125 nm. Cell uptake is analyzed by flow cytometry and reveals a higher uptake of PLLA particles compared to PMMA and PS particles. In competitive uptake studies two different types of particles are co-incubated with the HeLa cells; the results indicate a mutual influence of the particles on their uptake behavior. A reduced internalization of PLLA particles in the presence of PS particles is observed, although neither the co-incubation of PMMA and PLLA nor of PMMA and PS shows similar effect.     

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.     

Tracking of single fluorescent particles in three dimensions: use of cylindrical optics to encode particle position.

We present a novel optical technique for three-dimensional tracking of single fluorescent particles using a modified epifluorescence microscope containing a weak cylindrical lens in the detection optics and a microstepper-controlled fine focus. Images of small, fluorescent particles were circular in focus but ellipsoidal above and below focus; the major axis of the ellipsoid shifted by 90 degrees in going through focus. Particle z position was determined from the image shape and orientation by applying a peak detection algorithm to image projections along the x and y axes; x, y position was determined from the centroid of the particle image. Typical spatial resolution was 12 nm along the optical axis and 5 nm in the image plane with a maximum sampling rate of 3-4 Hz. The method was applied to track fluorescent particles in artificial solutions and living cells. In a solution of viscosity 30 cP, the mean squared distance (MSD) traveled by a 264 nm diameter rhodamine-labeled bead was linear with time to 20 s. The measured diffusion coefficient, 0.0558 +/- 0.001 micron2/s (SE, n = 4), agreed with the theoretical value of 0.0556 micron2/s. Statistical variability of MSD curves for a freely diffusing bead was in quantitative agreement with Monte Carlo simulations of three-dimensional random walks. In a porous glass matrix, the MSD data was curvilinear and showed reduced bead diffusion. In cytoplasm of Swiss 3T3 fibroblasts, bead diffusion was restricted. The water permeability in individual Chinese Hamster Ovary cells was measured from the z movement of a fluorescent bead fixed at the cell surface in response osmotic gradients; water permeability was increased by > threefold in cells expressing CHIP28 water channels. The simplicity and precision of this tracking method may be useful to quantify the complex trajectories of fluorescent particles in living cells.