Sampling efficiency of a single-cell capillary electrophoresis system.

Capillary electrophoresis (CE) combined with a laser-induced fluorescence (LIF) detection scheme is a powerful approach for single-cell analysis. For measurements requiring a high temporal resolution, CE-LIF is often combined with cell lysis systems based on pulsed lasers. Although extremely rapid, laser lysis has raised some concerns about the efficiency at which the cell contents are sampled. We have assembled a single-cell CE-LIF mounted on the stage of a microscope. This system was coupled with a nanosecond pulsed laser for cell lysis. We have analyzed green fluorescent protein (GFP) expressed in single mammalian cells and developed a novel approach to estimate the cell sampling efficiency (SE) based on the use of fluorescent calibration microspheres and flow cytometry. A significant advantage of this method is that it does not require any knowledge or assumption regarding the cell volume. We have evaluated the SE for different laser pulse energies (from 2 to 9 microJ) and two different pulse focal positions in the xy plane (0-10 microm from the center of the cell). We found the maximum SE at the lowest energy (2 microJ), with the pulse focused directly on the cell. We have demonstrated the utility of a novel method to measure the SE of a single-cell CE system. The measurements presented in this study indicate that rapid cell lysis with nanosecond lasers requires careful optimization of pulse parameters for maximum sampling of the cell contents.     

Three-color immunofluorescence analysis of mouse B-lymphocyte subpopulations

We have modified a fluorescence-activated cell sorter (FACS) to make three independent immunofluorescence measurements on each cell and used this system to study mouse B-lymphocyte subpopulations. An argon-ion laser (emitting at 488 nm) excites fluorescein- and phycoerythrin-labeled reagents, and a tunable dye laser charged with rhodamine 6G (emitting at 615 nm) excites an allophycocyanin-labeled reagent. We report simultaneous measurements of IgM, IgD, and the recently-defined mouse B lymphocyte antigens BLA-1 and BLA-2 on splenic lymphocytes of CBA/J mice and mice of the congenic strain CBA/N (which have an X-linked immunodeficiency [xid]). These data provide information on relationships among the B-cell populations in CBA/J "normal" mice and the defective CBA/N that could not be derived from one- or two-color immunofluorescent measurements. We believe this is the first use of allophycocyanin as an immunofluorescence label.     

Laminar flow cells for single-molecule studies of DNA-protein interactions

Microfluidic flow cells are used in single-molecule experiments, enabling measurements to be made with high spatial and temporal resolution. We discuss the fundamental processes affecting flow cell operation and describe the flow cells in use at present for studying the interaction of optically trapped or mechanically isolated, single DNA molecules with proteins. To assist the experimentalist in flow cell selection, we review the construction techniques and materials used to fabricate both single- and multiple-channel flow cells and the advantages of each design for different experiments.     

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.     

Single cell analysis using surface enhanced Raman scattering (SERS) tags

Fluorescence is a mainstay of bioanalytical methods, offering sensitive and quantitative reporting, often in multiplexed or multiparameter assays. Perhaps the best example of the latter is flow cytometry, where instruments equipped with multiple lasers and detectors allow measurement of 15 or more different fluorophores simultaneously, but increases beyond this number are limited by the relatively broad emission spectra. Surface enhanced Raman scattering (SERS) from metal nanoparticles can produce signal intensities that rival fluorescence, but with narrower spectral features that allow a greater degree of multiplexing. We are developing nanoparticle SERS tags as well as Raman flow cytometers for multiparameter single cell analysis of suspension or adherent cells. SERS tags are based on plasmonically active nanoparticles (gold nanorods) whose plasmon resonance can be tuned to give optimal SERS signals at a desired excitation wavelength. Raman resonant compounds are adsorbed on the nanoparticles to confer a unique spectral fingerprint on each SERS tag, which are then encapsulated in a polymer coating for conjugation to antibodies or other targeting molecules. Raman flow cytometry employs a high resolution spectral flow cytometer capable of measuring the complete SERS spectra, as well as conventional flow cytometry measurements, from thousands of individual cells per minute. Automated spectral unmixing algorithms extract the contributions of each SERS tag from each cell to generate high content, multiparameter single cell population data. SERS-based cytometry is a powerful complement to conventional fluorescence-based cytometry. The narrow spectral features of the SERS signal enables more distinct probes to be measured in a smaller region of the optical spectrum with a single laser and detector, allowing for higher levels of multiplexing and multiparameter analysis.     

Influence of cardiac flow rate on turbulent shear stress from a prosthetic heart valve

Elevated turbulent shear stresses associated with sufficient exposure times are potentially damaging to blood constituents. Since these conditions can be induced by mechanical heart valves, the objectives of this study were to locate the maximum turbulent shear stress in both space and time and to determine how the maximum turbulent shear stress depends on the cardiac flow rate in a pulsatile flow downstream of a tilting disk valve. Two-component, simultaneous, correlated laser velocimeter measurements were recorded at four different axial locations and three different flow rates in a straight tube model of the aorta. All velocity data were ensemble averaged within a 15 ms time window located at approximately peak systolic flow over more than 300 cycles. Shear stresses as high as 992 dynes/cm2 were found 0.92 tube diameters downstream of the monostrut, disk valve. The maximum turbulent shear stress was found to scale with flow rate to the 0.72 power. A repeatable starting vortex was shed from the disk at the beginning of each cycle.     

Flow cytometric studies during culture of Saccharomyces cerevisiae

Double-beam laser flow cytometry has been used to analyse samples from different Saccharomyces cerevisiae cultivation processes (batch and continuous culture). Using this technique, the biological parameters of cell size, and protein, RNA and DNA content can be determined. It is possible to study the budding behaviour from the cell size distribution as well as from DNA analysis. In oscillating continuous yeast cultures, flow cytometry studies were performed in addition to on-line NADH measurements. The effect of cell recycling on yeast metabolism was also the object of cytometric studies. Structured biological data can be obtained by this analytical technique, and the effect of different cultivation conditions on cell biology can be determined.     

High resolution dual laser flow cytometry.

Flow cytometers based on optical sensing utilize external light sources and fluorescent dyes to measure one or more specific components or properties of individual cells or subcellular particles in liquid suspension. To provide for independent excitation of two dyes used in double staining experiments we have constructed a high resolution flow cytometer that uses two laser beams to provide two wavelengths of excitation. These beams are separated spatially so that cells flow through them sequentially, with a time separation of about 20 musec. Since the dyes are excited sequentially their emission occurs at different times and their emission spectra may overlap without causing any difficulty in analysis. We have developed new light collection optics that permit up to four measurements to be made on each cell. This approach greatly increases the number of dye combinations that can be used in flow cytometry, thus removing a significant limitation of single illumination instruments.     

A generalized machine for automated flow cytology system design.

A general-purpose multiparameter flow cytophotometry system has been developed for use in the desgin of flow cytophotometers to perform specific tasks in automated cytology. Five separate measurement stations spaced along the axis of a capillary tube can be used to make up to eight optical measurements of individual cells flowing through the capillary. The system uses a broad-band arc source and can measure light scattered at various angles, light absorption by cell constituents and/or dyes and fluorescence of cell constituents and/or fluorochromes, excited directly and/or by energy transfer from neighboring molecules. High numerical aperture optics are used to maximize light-gathering capacity and minimize the effects of cell orientation and eccentricity of position in the fluid stream on measurements. A hard-wired preprocessor is used to detect the presence of cells and adjust sampling timing for changes in cell velocity; the electronic system also controls the gain of the detector photomultiplier tubes to compensate for background variations. Data acquistion and analysis are controled by a small general-purpose digital computer. The system has been used to develop a method and apparatus for blood cell counting and classification.     

Immunofluorescence measurement in a flow cytometer using low-power helium-neon laser excitation

Helium-neon lasers are economical and efficient light sources; their utility in flow cytometry to date has been limited by the lack of fluorescent probes that can be excited at 633 nm. Allophycocyanin (APC), a highly fluorescent phycobiliprotein, can be used as an antibody label and has spectral characteristics suitable for use with He-Ne lasers; we undertook to resolve whether a low-power (7 mW) He-Ne laser could provide sufficient excitation to permit flow cytometric detection of APC-labeled antibodies on cell surfaces. We made an APC conjugate of monoclonal antibody 4F2, which reacts with an antigen abundant on the surfaces of activated human T-lymphocytes; APC-4F2 was used to stain blood mononuclear cells that had been cultured with and without phytohemagglutinin (PHA). Cells so stained were examined in a flow cytometer with orthogonal illumination at 633 nm from a 7 mW He-Ne laser; antibody-bearing cells were detectable by fluorescence emission above 665 nm. Cells from the same cultures were stained with fluorescein-labeled 4F2 antibody and examined in a flow cytometer with argon ion laser excitation at 488 nm. Percentages of antibody-bearing cells determined from APC fluorescence and from fluorescein fluorescence were in good agreement. It thus appears that He-Ne lasers and APC-antibodies are usable for immunofluorescence measurements; the sensitivity attainable with this technique remains to be determined.     

An easy-to-use simulation program demonstrates variations in bacterial cell cycle parameters depending on medium and temperature

Many studies are performed on chromosome replication and segregation in Escherichia coli and other bacteria capable of complex replication with C phases spanning several generations. For such investigations an understanding of the replication patterns, including copy numbers of origins and replication forks, is crucial for correct interpretation of the results.Flow cytometry is an important tool for generation of experimental DNA distributions of cell populations. Here, a Visual Basic based simulation program was written for the computation of theoretical DNA distributions for different choices of cell cycle parameters (C and D phase durations, doubling time etc). These cell cycle parameters can be iterated until the best fit between the experimental and theoretical DNA histograms is obtained. The Excel file containing the simulation software is attached as supporting information.Cultures of Escherichia coli were grown at twelve different media and temperature conditions, with following measurements by flow cytometry and simulation of the DNA distributions. A good fit was found for each growth condition by use of our simulation program. The resulting cell cycle parameters displayed clear inter-media differences in replication patterns, but indicated a high degree of temperature independence for each medium. The exception was the poorest medium (acetate), where the cells grew with overlapping replication cycles at 42 °C, but without at the lower temperatures.We have developed an easy-to-use tool for determination of bacteria's cell cycle parameters, and consequently the cells' chromosome configurations. The procedure only requires DNA distribution measurements by flow cytometry. Use of this simulation program for E. coli cultures shows that even cells growing quite slowly can have overlapping replication cycles. It is therefore always important not only to assume cells' replication patterns, but to actually determine the cell cycle parameters when changing growth conditions.     

A new multiparameter flow cytometer: optical and electrical cell analysis in combination with video microscopy in flow

BACKGROUND: Flow cytometers, which are commercially available, do not necessarily meet all demands of actual biomedical research. This is the case for the investigation of mechanisms involved in cell volume regulation, which requires electrical volume measurement and ratiometric multichannel fluorescence analysis for the simultaneous assessment of different physiologic parameters (intracellular pH and the intracellular concentration of calcium ions, etc). METHODS AND RESULTS: We describe the construction of a new nonsorting flow cytometer designed for the simultaneous acquisition of seven parameters including fluorescence signals, forward and perpendicular light scatter, cell volume according to the electrical Coulter principle, and flow cytometric imaging. The instrument is equipped with three different light sources. A tunable argon-ion laser generates efficient excitation of the most standard fluorescent probes in the visible spectral range, and an arc lamp provides the means for ultraviolet excitation at low cost. Because of the spatial filtering by the excitation and detection optics, two independent sets of dual fluorescence measurements can be performed, a prerequisite for flexible ratiometric fluorescence analysis. A flow video microscope integrated into the optical system optionally generates either brightfield or phase images of selected flowing particles. Only particles whose individual datasets meet predefined gating conditions are imaged in real time. To avoid smear effects, the motion of the object to be imaged (speed approximately 8 m/s) is frozen on the target of a CCD camera by flash illumination. For this purpose, a high radiance gas discharge lamp with 25-mJ electric pulse energy provides an illumination time of 18 ns (full width half maximum). Test results obtained from latex spheres and cells are shown. CONCLUSIONS: Test results indicate that our instrument can perform Coulter measurements in combination with flexible optical analysis. Moreover, integration of an adapted video microscope into a flow cytometer is an approach to overcome the gap between flow and image cytometry.     

Real-time cortical cerebral blood flow follow-up in conscious,freely moving rats by laser Doppler flowmetry

This article describes a laser Doppler flowmetry (LDF) system that enables repeated measurements and thereby long-term followup of cortical cerebral blood flow (CBF) in awake and freely moving rats. The system consists of a specially designed flow probe adapter, a flow probe connector, and a LDF flow probe, which may thereby rotate through its own axis. During the experiment, the flow adapter is permanently mounted onto the rat's skull bone. A thin layer of skull bone is left intact at the site for cortical CBF measurements. The probe connector and the flow probe may be repeatedly detached and remounted to the adapter, which allows for cortical cerebral blood flow recording from exactly the same anatomical location. The laser Doppler flowmetry system enables stable cortical CBF recordings in the conscious rat while it moves freely in a bowl cage.     

Evaluation of a green laser pointer for flow cytometry.

Flow cytometers typically incorporate expensive lasers with high-quality (TEM00) output beam structure and very stable output power, significantly increasing system cost and power requirements. Red diode lasers minimize power consumption and cost, but limit fluorophore selection. Low-cost DPSS laser pointer modules could possibly offer increased wavelength selection but presumed emission instability has limited their use. A $160 DPSS 532 nm laser pointer module was first evaluated for noise characteristics and then used as the excitation light source in a custom-built flow cytometer for the analysis of fluorescent calibration and alignment microspheres. Eight of ten modules tested were very quiet (RMS noise < or = 0.6% between 0 and 5 MHz). With a quiet laser pointer module as the light source in a slow-flow system, fluorescence measurements from alignment microspheres produced CVs of about 3.3%. Furthermore, the use of extended transit times and < or =1 mW of laser power produced both baseline resolution of all 8 peaks in a set of Rainbow microspheres, and a detection limit of <20 phycoerythrin molecules per particle. Data collected with the transit time reduced to 25 micros (in the same instrument but at 2.4 mW laser output) demonstrated a detection limit of approximately 75 phycoerythrin molecules and CVs of about 2.7%. The performance, cost, size, and power consumption of the tested laser pointer module suggests that it may be suitable for use in conventional flow cytometry, particularly if it were coupled with cytometers that support extended transit times.     

Utility of light scatter in the morphological analysis of sperm.

We were able to differentiate the morphologically diverse sperm nuclei of four animal species by using an Ortho flow cytometer to detect the forward light scatter from a red (helium-neon) laser. Cytograms depicting the axial light loss and forward red scatter signals revealed unique, but reproducible, sigmoid distributions that reflected not only interspecies differences in shape and size, but variations in particle refractive index and orientation within the flow cell at the time of analysis. Consequently, we were able to use regional gating of the light scatter cytogram to minimize the influence of orientation on the resolution of the fluorescence signal. We also observed that sperm enlarging as a result of chemically induced decondensation exhibit over time a biphasic shift (increase, then decrease) in light scatter at a species-dependent rate. These results suggest that, without any special adaptations to the flow cytometer, light-scatter parameters can be used to discriminate morphologically different sperm, to enhance the resolution of fluorescence measurements that may otherwise be confounded by variability in radial orientation, and to detect alterations in the rate of a biochemical/biophysical process such as decondensation.     

Imaging technique implemented in CellTracks system

BACKGROUND: We developed the CellTracks cell analysis system that, similar to flow cytometry, yields multiparameter information by which the cells can be differentiated. We describe the implementation of a laser scanning imaging method in the system. Image analysis of the cells improves the specificity of cell classification, especially in cases where the particular cells are found relatively infrequently and one has to discriminate between artifacts and real events. METHODS: Fluorescent images of immunomagnetically labeled and aligned cells are obtained by passing the cells through a laser focus. The laser focus is smaller than the objects and subsequent frames captured by a regular surveillance CCD camera with a frame grabber board represent different parts of the cells. Complete images of the cells are constructed by shifting each image with respect to each other and adding individual pixel values. RESULTS: The power of combining a fluorescent image with multiparametric data is demonstrated by imaging fluorescent and magnetically labeled beads and cells. The image gives additional information about the dye distribution across the objects. Changes in dye distribution as a function of time were observed in leukocytes labeled with the red fluorescent label, Oxazine750, which are imaged at different time intervals. CONCLUSIONS: An imaging technique implemented in the CellTracks system provides high-resolution fluorescent images of events previously identified by the system. The images of the fluorescent cells enhance the ability to classify rare events.     

Non-destructive analysis of the nuclei of transgenic living cells using laser tweezers and near-infrared raman spectroscopic technique

Transgenic cell lines of loblolly pine （Pinus taeda L.） were analyzed by a compact laser-tweezers-Raman-spectroscopy （LTRS） system in this investigation. A low power diode laser at 785 nm was used for both laser optical trapping of single transgenic cells and excitation for near-infrared Raman spectroscopy of the nuclei of synchronized cells, which were treated as single organic particles, at the S-phase of the cell cycle. Transgenic living cells with gfp and uidA genes were used as biological samples to test this LTRS technique. As expected, different Raman spectra were observed from the tested biological samples. This technique provides a high sensitivity and enables real-time spectroscopic measurements of transgenic cell lines. It could be a valuable tool for the study of the fundamental cell and molecular biological process by trapping single nucleus and by providing a wealth of molecular information about the nuclei of cells.     

Development of microscopic systems for high-speed dual-excitation ratiometric Ca2+ imaging

For quantitative measurements of Ca(2+) concentration ([Ca(2+)]), ratiometric dyes are preferable, because the use of such dyes allows for correction of uneven loading or partitioning of dye within the cell as well as variations in cell thickness. Although dual-excitation ratiometric dyes for measuring [Ca(2+)], such as Fura-2, Fura-Red, and ratiometric-pericam, are widely used for a variety of applications, it has been difficult to use them for monitoring very fast Ca(2+) dynamics or Ca(2+) changes in highly motile cells. To overcome this problem, we have developed three new dual-excitation ratiometry systems. (1) A system in which two laser beams are alternated on every scanning line, allowing us to obtain confocal images using dual-excitation ratiometric dyes. This system increases the rate at which ratio measurements can be made to 200 Hz and provides confocal images at 1-10 Hz depending on the image size. (2) A truly simultaneous dual-excitation ratiometry system that used linearly polarized excitation light and polarization detection, allowing us to obtain ratiometric images without any time lag. This system, however, is based on statistical features of the fluorescence polarization and is limited to samples that contain a large number of fluorophores. In addition, this method requires complicated calculations. (3) An efficient, nearly simultaneous dual-excitation ratiometry system that allows us to rapidly switch between two synchronized excitation-detection components by employing two high-power light-emitting diodes (LEDs) and two high-speed liquid crystal shutters. The open/close operation of the two shutters is synchronized with the on/off switching of the two LEDs. This system increases the rate at which ratio measurements are made to 1 kHz, and provides ratio images at 10-100 Hz depending on the signal intensity.     

A means for orienting flat cells in flow systems.

Flattened cells, such as red blood cells, epithelial cells, and sperm of many species, cause problems for fluorescence-activated cell analysis and sorting machines because the flow systems of such devices are unable to control the orientation of these cells as they flow past the detectors. For this reason, the fluorescence or scattered light measurements for identical cells may vary greatly. A flow geometry is here described that orients flat cells in a coaxial flow system so that each cell presents the same aspect to the observation device. A wedge-shaped exit on the sample injection tube in a coaxial flow system is sufficient to produce the desired orientation effect when used with low sample flow rates. Data is presented showing the effect of orientation of fixed chicken erythrocytes on histograms of small forward-angle light-scattering measurements.