Design of flow chamber with electronic cell volume capability and light detection optics for multilaser flow cytometry

A multibeam optical detection system has been developed with a high optical efficiency, achieved through a reduction in the number of optical interfaces employed in the system. This reduction is made possible by a combination of employing simple lenses, gluing the objective lens directly upon the face of the flow cuvette and the extraction of only one fluorescence signal from each laser beam. A modified flow chamber is also described that includes fluidic resistance elements for the elimination of most of the electric shielding normally associated with electronic cell volume measurements.     

Flow cytometry for immunology

Improvement in our knowledge in cellular biology is largely related to the use of new tools in quantitative cytology. Among them, flow cytometry was developed with numerous applications in the field of immunology including fundamental and applied research. Since its early beginning it has been associated with monoclonal antibodies to identify immuno-competent cells, to quantify changes in expression of surface determinants, to separate cells subsets prior to the test of their functional properties. Major advances gained using either single or dual-laser systems, multicolour fluorescence and computer facilities for multi-parametric analysis. Using this methodology it was possible to correlate analysis of cell cycle phases and membrane antigens expression. Applications have been developed for the analysis of new drugs in vitro, the evaluation of immunomodulating treatment and for clinical investigations.     

A phototube connecting digital cameras with light optic microscopes

We present here a phototube model making possible connection of a digital camera with light optic microscopes in order to obtain images of microobjects and for their further computer treatment. The advantage of this model is simplicity of its manufacturing and small required expenses as well as an increase in information density for microobject studies. This phototube has been covered by a patent for a useful model N2 48228 registered in the Public Register of RF on September 27, 2005.     

Comparison of flow cytometry and epifluorescence microscopy for counting bacteria in aquatic ecosystems.

Flow cytometry was used to count bacterial cells from diverse origins: one strain of E. coli, one sample of lake water, and 18 samples of estuary water. To verify the accuracy and the precision of this technique, total bacteria counts made by flow cytometry were compared with counts by direct observation using epifluorescence microscopy. The results of this study showed that flow cytometry was a reliable technique for counting a mixture of bacteria in samples from aquatic ecosystems.     

Flow cytometry for the development of biotechnological processes with microalgae

The current interest in microalgae as a sustainable source of next generation biofuels and other valuable substances is driving exploration of their use as unique biotechnological production systems. To design and optimise appropriate production strategies, the behaviour of particular microalgal species should be well characterised under different culture conditions. Thus, flow cytometric (FCM) methods, which are already well established in environmental and toxicological studies of microalgae, are also useful for analysing the physiological state of microalgae, and have the potential to contribute to the rapid development of feasible bioprocesses. These methods are commonly based on the examination of intrinsic features of individual cells within a population (such as autofluorescence or size). Cells possessing the desired physiological or morphological features, which are detectable with or without fluorescent staining, are counted or isolated (sorted) using an FCM device. The options for implementation of FCM in the development of biotechnological processes detailed in this review are (i) analysing the chemical composition of biomass, (ii) monitoring cellular enzyme activity and cell viability, and (iii) sorting cells to isolate those overproducing the target compound or for the preparation of axenic cultures.     

Flow cytometers using optical waveguides in place of lenses for specimen illumination and light collection

Optical waveguides represent alternatives to lenses for delivery of light to, and collection of light from, small regions of space such as the observation point(s) of a flow cytometer. The flow chamber and directly associated illumination and collection optics of a flow cytometer can be built using fiber optic waveguides in fixed positions or as an integrated optical assembly incorporating deposited or implanted waveguides, providing illumination and collection geometries and efficiencies comparable to conventional designs, with the advantage that the optical waveguide design, once assembled, never again requires alignment of the optics. A prototype apparatus in which fiber optics were used to illuminate and collect light from a point inside a thin-walled, round quartz capillary was observed to measure fluorescence of polystyrene spheres with precision close to that obtained from a conventional flow cytometer using the same laser source. The design of the fiber optic system is readily adaptable to construction of multiple illumination beam instruments; other optical waveguide flow cytometer designs that dispense with the capillary share this advantage and can also provide orifices for electronic volume sensing and for jetting prior to droplet sorting.     

Flow cytometry for high-throughput, high-content screening

Flow cytometry is a mature platform for quantitative multi-parameter measurement of cell fluorescence. Recent innovations allow up to 30-fold faster serial processing of bulk cell samples. Homogeneous discrimination of free and cell-bound fluorescent probe eliminates wash steps to streamline sample processing. Compound screening throughput may be further enhanced by multiplexing of assays on color-coded bead or cell suspension arrays and by integrating computational techniques to create smaller, focused compound libraries. Novel bead-based assay systems allow studies of real-time interactions between solubilized receptors, ligands and molecular signaling components that recapitulate and extend measurements in intact cells. These new developments, and its broad usage, position flow cytometry as an attractive analysis platform for high-throughput, high-content biological testing and drug discovery.     

Quantitative intercomparison of transmission electron microscopy,flow cytometry,and epifluorescence microscopy for nanometric particle analysis

Nanometric biological particles such as viruses have received increased attention in a wide range of scientific fields. Evaluation of viral contributions to environmental processes and the use of viruses in medical applications such as gene therapy require viruses to be routinely and accurately enumerated. There are a variety of existing techniques for counting viruses, namely, plaque assays, transmission electron microscopy (TEM), epifluorescence microscopy (EFM), and flow cytometry (FCM); each has advantages and disadvantages. While there have been attempts to intercompare some of these techniques to determine the most effective means to count viruses, no previous study used a technique-independent standard for quantitative comparison of collection efficiency, accuracy, and precision. In this work, polystyrene nanospheres were used as standards for the intercomparison of performance characteristics for TEM, EFM, FCM, as well as a custom-built flow cytometer (the Single Nanometric Particle Enumerator, SNaPE). EFM and SNaPE exhibited the highest degree of accuracy and precision, with particle concentrations deviating < or =5% from true and relative errors less than half that of TEM, EFM and SNaPE are also significantly more time and cost efficient than TEM.     

Violet laser diodes as light sources for cytometry

BACKGROUND: Violet laser diodes have recently become commercially available. These devices emit 5-25 mW in the range of 395-415 nm, and are available in systems that incorporate the diodes with collimating optics and regulated power supplies in housing incorporating thermoelectric coolers, which are necessary to maintain stable output. Such systems now cost several thousand dollars, but are expected to drop substantially in price. Materials and Methods A 4-mW, 397-nm violet diode system was used in a laboratory-built flow cytometer to excite fluorescence of DAPI and Hoechst dyes in permeabilized and intact cells. Forward and orthogonal light scattering were also measured. RESULTS: DNA content histograms with good precision (G(0)/G(1) coefficient of variation 1.7%) were obtained with DAPI staining; precision was lower using Hoechst 33342. Hoechst 34580, with an excitation maximum nearer 400 nm, yielded the highest fluorescence intensity, but appeared to decompose after a short time in solution. Scatter signals exhibited relatively broad distributions. CONCLUSIONS: Violet laser diodes are relatively inexpensive, compact, efficient, and quiet light sources for DNA fluorescence measurement using DAPI and Hoechst dyes; they can also excite several other fluorescent probes.     

Flow cytometry of Escherichia coli for process monitoring

A laser flow cytometer was used to study different Escherichia coli populations under various cultivation conditions. A host strain E. coli 5K was analyzed for cell size, protein and DNA-content during continuous cultivation. Also, a recombinant E. coli 5K(pHM12) strain (used for the intracellular production of penicillin-G acylase) was studied in regard to gene expression using different cytometric techniques. An argon ion laser (30 mW) and a 100 W high-pressure mercury lamp were used as light source in the cytometer. A new fluorogenic staining technique for intracellular penicillin-G acylase is described.Recombinant E. coli temperature sensitive cells were analyzed for intracellular fusion protein production due to temperature induction.     

The development of a cryopreservation method suitable for a large cyanobacteria collection

Laboratories worldwide working with cyanobacteria have created culture collections to carry out related studies. However, the lack of manpower (especially after the studies were finished), maintenance costs and proper preservation methods often results in the loss of research materials and a waste of isolation effort. Several parameters are generally considered very important in cryopreservation, including the choice of the cryoprotectant, cryoprotectant concentration, freezing rate, physiological status of the culture and thawing procedure. This makes it very difficult to establish universal guidelines for cyanobacteria cryopreservation. Herein, we present a cryopreservation method suitable for a range of strains, using two cryoprotectants (methanol and dimethyl sulfoxide at a final concentration of 5 and 3 %, respectively) along with a combined vital staining and reproductive viability criteria for the post-thawing recovery. The obtained results are very encouraging as more than 83 % of tested cyanobacteria were amenable for cryopreservation, 80 % of strains (111 in total) showing more than 90 % recovery with at least one of the cryoprotectants used.     

Flow cytometry of plant cells with applications in large-scale bioprocessing

In recent years, there has been a significant upsurge in the application of flow cytometry to plant cells and plant cell cultures. As well as a range of uses in plant biology, flow cytometry offers many advantages for monitoring plant cell cultures used in large-scale bioprocessing operations. This review summarizes the current status of the field, concentrating on methods for DNA measurement and multiparameter cell cycle analysis. Techniques for screening and selection of elite cell lines with high productivity of secondary metabolites are also addressed.     

A cytochemical method for fixation and staining of lipids in frozen-dried mouse pancreas for study with light and electron microscopes

Summary A method is described for the cytochemical identification of lipids in acinar cells of the pancreas based chiefly on reactions of their carboxyl ester linkages and their double bonds. The method involves the reaction in vacuo of certain amine and hydrazine vapors with lipids in the solid state. The method is useful for studies with the light and electron microscopes.This research was supported in part by grants from the Public Health Service (GM 08328) and the Commonwealth Fund.I have been guided throughout by the perceptive advice of Professor Herbert S. Anker, Department of Biochemistry, University of Chicago. I am indebted also to the tireless work and support of Mrs. Faustina Manelis and Mrs. Elizabeth Vilkas.Dedicated to Professor Friedrich Wassermann with admiration and affection on the occasion of his eightieth birthday.     

Developing a common bean core collection suitable for association mapping studies

Because of the continuous introduction of germplasm from abroad, some collections have a high number of accessions, making it difficult to explore the genetic variability present in a germplasm bank for conservation and breeding purposes. Therefore, the aim of this study was to quantify and analyze the structure of genetic variability among 500 common bean accessions to construct a core collection. A total of 58 SSRs were used for this purpose. The polymorphism information content (PIC) in the 180 common bean accessions selected to compose the core collection ranged from 0.17 to 0.86, and the discriminatory power (DP) ranged from 0.21 to 0.90. The 500 accessions were clustered into 15 distinct groups and the 180 accessions into four distinct groups in the Structure analysis. According to analysis of molecular variance, the most divergent accessions comprised 97.2% of the observed genetic variability present within the base collection, confirming the efficiency of the selection criterion. The 180 selected accessions will be used for association mapping in future studies and could be potentially used by breeders to direct new crosses and generate elite cultivars that meet current and future global market needs.