Attogram detection limit for aqueous dye samples by laser-induced fluorescence.

A modified flow cytometer has been used to detect attogram quantities of aqueous rhodamine 6G by laser-induced fluorescence analysis. A detection limit of 28 attograms (35,000 molecules) was obtained, nearly two orders of magnitude better than earlier measurements. The detection limit in concentration units was 1.4 x 10(-13) mole per liter. During the 1-second measurement period, the total volume sampled was 0.42 microliter. On average, only half a rhodamine 6G molecule was present in the 6-picoliter probed volume.

Light scattering with stream-in-air flow systems.

Both forward angle and 90 degrees light-scattering measurements have been used for cell sizing with stream-in-air flow systems with very little theoretical base for the measurements. Mie theory calculations are compared with measurements on plastic microspheres. Detector response for homogeneous spheres is shown to be sensitive to refractive index.

Laser flow cytometric light scatter and fluorescence pulse width and pulse rise-time sizing of mammalian cells.

In laser flow cytometry, an increasingly popular technique of analytical cytology, quantitative measurements of interest include cell and nuclear diameters. Electronic circuitry for a new cell sizing technique has been developed which measured the time that signal pulses from either fluorescence or light scatter sensors exceed a preset constant fraction of the peak signal amplitude (pulse width) or the time that it takes a signal to rise between constant fractions of the peak signal amplitude on the rising side of the pulse (pulse rise-time). These pulse width or pulse rise-time measurements were related to cell or nuclear diameters and were used in combination to determine nuclear size to cell size ratios. This method of sizing was found to be independent of fluorescent or light-absorbing stain intensity, linearly related to cell or nuclear diameter, and capable of resolving small diameter differences.

Flow microfluorometric quantitation of the blastogenic response of lymphocytes.

A method for quantitating the specific stimulation of peripheral lymphocytes has been developed using the techniques of flow microfluorometry. Peripheral bovine lymphocytes were collected and specifically stained for deoxyribonucleic acid (DNA) content using a low-salt propidium iodide procedure. Flow microfluorometry was used to determine, on the basis of DNA content, the percentage of cells in a population that was stimulated. Extremely uniform staining of the lymphocytes (coefficient of variation of less than 2%) provides a high resolution between proliferating and nonproliferating cells. The method provides a rapid, highly repoducible technique for determing the fraction of lymphocytes stimulated in response to tuberculin antigens based on an increase in cellular DNA content. Specific and nonspecific stimulation by a defined antigen can be measured and resolved.

High-speed DNA sequencing: an approach based upon fluorescence detection of single molecules.

We are developing a laser based technique for the rapid sequencing of large fragments (approximately 40 kb) of DNA based upon the detection of single, fluorescently tagged nucleotides cleaved from a single DNA fragment. We have demonstrated significant progress on several of the important steps of this technique. The projected rate of sequencing is several hundred bases per second which is orders of magnitude faster than existing methods. Once developed, this technology could be utilized by investigators for rapid sequencing of genetic material from virtually any source.

Molecular cytometry.

The first flow cytometric analyses were of total cellular DNA or protein content. The advent of labeled antibodies, first polyclonal and then monoclonal, led to the analysis of cellular content of specific antigen molecules and a myriad of immunological applications. Although, strictly speaking, it is incorrect to use the word "cytometry" for anything that does not refer to cellular measurements, the term has been used for a variety of non-cellular measurements such as chromosome analysis and solution molecular measurements. In recent years, there has been an increase in the development of methods to analyze molecules and their properties in solution. These analyses have ranged from ensemble measurements, primarily fluorescence-based immunoassays, to single molecule detection with applications in DNA analysis.

New flow cytometric technologies for the 21st century.

The envelope that defines the limits within which flow cytometry was developed is being rapidly expanded. For example: detection sensitivity has been extended to single molecules, the size range of "particle" analysis now extends from DNA fragments to plankton (1,000.+ microns), cell and chromosome sorting rates are being increased dramatically by using inactivation procedures (50,000 per second versus 2,000 per second), rapid kinetic flow cytometry enables real-time analysis of molecular assembly and cell function in the sub-second time domain, the lifetime of a fluorochrome bound to a single cell can be measured with nsec precision, and classical karyotype information (cell to cell heterogeneity) can be determined in a flow based system. These frontiers have greatly expanded the range of new and exciting flow cytometric based biomedical applications. New enabling technologies have provided the means to measure DNA cleavage by the structure-specific nuclease, human Flap Endonuclease (FEN-1), in the 300 msec time frame. Phase sensitive measurements and fluorescence lifetime are proving to be major advances for understanding molecular environments that change with, for example, the process of apoptosis. The ability to detect single fluorescent molecules has been applied to the analysis of DNA fragments obtained from enzymatic digestion of lambda DNA. This technology is being used to rapidly and very accurately size DNA fragments for the human genome project. Optical chromosome selection is a faster, better, less complex approach to chromosome sorting. This method is based on the induction of specific damage to the DNA of selected chromosomes. Lastly, the miniaturization of a single cell fractionator has made it possible to perform single cell flow cytogenetics.

Droplet sorting of large particles.

The systematics of droplet formation conditions for orifices with diameters up to 200 micron are described. Sorting recovery experiments indicate that particles up to 44 micron in diameter can be recovered by charged droplet deflection of two drops with at least 75% recovery. By reducing the jet velocity, a deflection of greater than 1 cm was obtained for all droplet sizes.

Cell cycle kinetics of Chinese hamster (CHO) cells treated with the iron-chelating agent picolinic acid.

Spontaneously transformed (tumorigenic) Chinese hamster cells (line CHO) do not exhibit picolinic acid-sensitive G1 and G2 cell cycle arrest points observed in normal and virus-transformed cells. Rather, picolinic acid arrests CHO cells in S phase only and produces culture growth behaviour similar to that produced by hydroxyurea. Prolonged treatment with picolinic acid permits a slow but significant traverse of cells through S phase. Thus, like hydroxyurea, picolinic acid is not a useful agent for synchronizing exponential CHO cells, but it can be used to resynchronize cultures in early S phase if a previous synchronization procedure (such as isoleucine deprivation) is used. The iron chelating properties of picolinic acid, and the similarities of its effects on cultured cells to those of hydroxyurea and the iron-chelating drug desferrioxamine, suggest that picolinic acid inhibits DNA synthesis by interfering with the iron-dependent production of a stable free organic radical which is essential for the ribonucleotide reductase formation of deoxyribonucleotides.

An improved sum-of-normals technique for cell cycle distribution analysis of flow cytometric DNA histograms.

An improved method for determining the fraction of cells in the G1, S and G2 + M phases of the life cycle, from DNA distributions of S phase rich cultures, is presented. Picolinic acid synchronized cell cultures were used to demonstrate the usefulness of the technique for the cell cycle distribution analysis. The algorithm presented quantifies DNA histograms from FCM analyses using an extension of the previously reported 'sum of discrete normal curves' technique. It alleviates problems encountered with earlier methods by extending the contribution of the S phase cells to the distribution under the G1 and G2 + M peaks; at the same time it allows more of the descriptive parameters to be determined from the individual histograms. The fractions of cells in the G1, S, and G2 + M phases of the life cycle obtained by this method compare favourably with autoradiographic analyses and other analytical results.

Modular electronics for flow cytometry and sorting: the LACEL system.

LACEL is newly developed, fast, general-purpose data acquisition and processing system for flow cytometric applications. The system's modular electronics allows flexibility in system configurations. The system can process as many as eight input analog parameters and can transfer 16-bit words between the user's electronics and the computer with standard input/output interfaces. The system's 8-fold coincidence logic capability can be set to operate with the noncoincidental timing that may occur in multiparameter flow measurements. As many as four parameters can be used to establish amplitude and timing criteria for each of two sorting directions. Two experiments can be on line with the computer at one time.

Techniques for flow cytometer alignment.

This unit provides essential knowledge for correctly using any flow cytometer to ensure that data collected are accurate and reliable. Two levels of system alignment are described: routine alignment checks and complete alignment, both of which can be applied to a variety of instrument configurations.

Bacteria genome fingerprinting by flow cytometry.

BACKGROUND: A flow cytometry-based, ultrasensitive fluorescence detection technique has been developed that demonstrates unique advantages in the analysis of large DNA fragments over the currently most widely used technology, pulsed-field gel electrophoresis (PFGE). The technique described herein is used to characterize the restriction fingerprints of the bacteria genome Staphylococcus aureus in this study. METHODS: The isolation of the bacterial genomic DNA and the subsequent complete digestion by a restriction endonuclease were performed inside an agarose plug. Electroelution was used to move the DNA fragments out-of the agarose plug into a solution containing low concentrations of spermine and spermidine, added to stabilize the large DNA fragments. DNA was stained with the bisintercalating dye thiazole orange homodimer (TOTO-1) and subsequently introduced into our ultrasensitive flow cytometer from a capillary. RESULTS: Individual DNA fragments up to 351 kbp were successfully handled and sized. The histograms of the burst sizes were generated from signals associated with individual fragments in <7 min with <2 pg of DNA. The sizing accuracy was better than 98%. In contrast, standard PFGE takes approximately 20 h and requires approximately 1 microg of DNA with a sizing accuracy of approximately 90%. CONCLUSIONS: With the demonstrated success and advantages, our approach has the potential of being applied to fast, accurate bacteria species and strain identification.

Amplified flow-cytometric separation-free fluorescence immunoassays.

An equilibrium-type competitive-binding fluorescence immunoassay with high sensitivity and excellent precision is described that obviates separation of free from bound label. In the assay relatively large (10 microns diameter) antibody-coated non-fluorescent particles and very small (0.10 micron diameter) antigen-coated fluorescent latex particles are used. Soluble nonlabeled antigen competes with antigen on the microspheres for antibody binding sites on the larger spheres. After equilibrium is attained, the fluorescence distribution of 5000 of the large spheres is measured in a flow cytometer. The mean values for the fluorescence distribution obtained from samples containing known concentrations of soluble antigen are used to construct a standard displacement curve. In a prototype assay for the antigen horseradish peroxidase, a sensitivity of 10(-12) mol/L has been achieved. Undiluted serum can be assayed without loss of sensitivity. Preliminary experiments also indicate that double-antibody "sandwich"-type assays of very high sensitivity (10(-14) mol/L) are also possible when this dual-sphere concept is exploited.