How should the optical tweezers experiment be used to characterize the red blood cell membrane mechanics?

Stretching red blood cells using optical tweezers is a way to characterize the mechanical properties of their membrane by measuring the size of the cell in the direction of the stretching (axial diameter) and perpendicularly (transverse diameter). Recently, such data have been used in numerous publications to validate solvers dedicated to the computation of red blood cell dynamics under flow. In the present study, different mechanical models are used to simulate the stretching of red blood cells by optical tweezers. Results first show that the mechanical moduli of the membranes have to be adjusted as a function of the model used. In addition, by assessing the area dilation of the cells, the axial and transverse diameters measured in optical tweezers experiments are found to be insufficient to discriminate between models relevant to red blood cells or not. At last, it is shown that other quantities such as the height or the profile of the cell should be preferred for validation purposes since they are more sensitive to the membrane model.     

Cytomat-R: a computer-controlled multiple laser source multiparameter flow cytophotometer system.

A multiple illumination wavelength multiparameter flow cytophotometer system, using laser sources and controlled by a small, general-purpose digital computer, has been produced for use in the development of new flow cytometric techniques. Three different laser wave-lengths can be used simultaneously to illuminate different regions of the flow chamber; as many as five measurements of light scattering at various angles, extinction, and fluorescence at one or more wavelengths can be made at each illuminated station. Cells in suspension may be examined at rates of 1000 cells/sec, with seven correlated optical measurements being recorded for each cell. A library of programs for data manipulation and statistical analysis make it possible to use the system to develop and implement cell characterization, counting and classification procedures for basic and clinical research applications.     

Identification of cell asymmetry and orientation by light scattering.

Light scattering from chicken red blood cells has been used as a model system to identify the asymmetry of cells. The histogram for forward angle light scattering for these cells is bimodal, the signal size being dependent on the cell orientation. A dual orthogonal scatter system is used to conclusively demonstrate this orientational variation in signal. A third scattering system, using a single incident beam with two orthogonal detectors, is used to further characterize the orientational variation of the scatter signal. In this third system it is shown that the signal in a detector set 90 degrees from the incident beam collects light reflected from the cell surface. The optical selection of cells in specific orientations using these systems may circumvent the need to physically orient cell in flow systems.     

Lateral view flow system for studies of cell adhesion and deformation under flow conditions

Physical interactions between circulating cells and the vascular wall play a central role in inflammation, metastasis, atherosclerosis, and therapeutic cell delivery. Unfortunately, traditional in vitro flow assays cannot be used to visualize the details of cell-surface interactions in blood flow because of inappropriate geometry and the poor penetration of light in erythrocyte solutions. To overcome these obstacles, we have developed an agarose-cast cylindrical vessel system to examine the profiles of cells interacting with surfaces under flow conditions. This design allows observation and quantification of cell deformation as cells adhere to surfaces under dynamic flow conditions without modifying the microscope or optical path. Furthermore, our flow system is uniquely suited for monitoring the profiles of adherent leukocytes deforming in response to erythrocyte suspension flow. We have used this flow system to study the role of erythrocytes in leukocyte-substrate interactions. Our results show that the cell deformation index (the ratio of the cell length to cell height) is higher in erythrocyte solutions compared to erythrocyte-free saline. This novel lateral view flow system provides a powerful technique for visualizing and quantifying the morphological changes of cells in contact with substrates exposed to shear stress.     

Identification and isolation of subpopulations of pleural cells by multiparameter flow cytometry

Multiparameter flow cytometry was used to identify and sort subpopulations of cells from pleural cell populations harvested from the rat without employing special stains or fluorochrome-labeled monoclonal antibodies. Cell parameters measured included electronic volume, axial light loss, 90° light scatter, and blue autofluorescence. Various bivariate combinations of these parameters were used to distinctly resolve pleural macrophages, eosinophils, mast cells, and lymphocytes. These subpopulations were separately sorted viably according to their unique electrooptical phenotypic characteristics in>90% purity. Our multiparameter flow cytometric approach, accordingly, provides a means by which pleural cell subpopulations may be easily obtained for subsequent in vitro study. Moreover, the general strategy for identifying and isolating these subpopulations may be usefully extended to the identification and isolation of subpopulations of cells occurring in other complex cell mixtures.     

Identification and isolation of subpopulations of pleural cells by multiparameter flow cytometry

Multiparameter flow cytometry was used to identify and sort subpopulations of cells from pleural cell populations harvested from the rat without employing special stains or fluorochrome-labeled monoclonal antibodies. Cell parameters measured included electronic volume, axial light loss, 90 degrees light scatter, and blue autofluorescence. Various bivariate combinations of these parameters were used to distinctly resolve pleural macrophages, eosinophils, mast cells, and lymphocytes. These subpopulations were separately sorted viably according to their unique electrooptical phenotypic characteristics in greater than 90% purity. Our multiparameter flow cytometric approach, accordingly, provides a means by which pleural cell subpopulations may be easily obtained for subsequent in vitro study. Moreover, the general strategy for identifying and isolating these subpopulations may be usefully extended to the identification and isolation of subpopulations of cells occurring in other complex cell mixtures.     

High-density photoautotrophic algal cultures: design, construction, and operation of a novel photobioreactor system

A photobioreactor system has been designed, constructed and implemented to achieve high photosynthetic rates in high-density photoautotrophic algal cell suspensions. This unit is designed for efficient oxygen and biomass production rates, and it also can be used for the production of secreted products. A fiber-optic based optical transmission system that is coupled to an internal light distribution system illuminates the culture volume uniformly, at light intensities of 1.7 mW/cm(2) over a specific surface area of 3.2 cm(2)/cm(3). Uniform light distribution is achieved throughout the reactor without interfering with the flow pattern required to keep the cells in suspension. An on-line ultrafiltration unit exchanges spent with fresh medium, and its use results in very high cell densities, up to 10(9) cells/mL [3% (w/v)] for eukaryotic green alga chlorella vulgaris. DNA histograms obtained form flow cytometric analysis reveal that on-line ultrafiltration influences the growth pattern. Prior to ultrafiltration the cells seem to have at a particular point in the cell cycle where they contain multiple chromosomal equivalents. Following ultrafiltration, these cells divide, and the new cells are committed to division so that cell growth resumes. The Prototype photobioreactor system was operated both in batch and in continuous mode for over 2 months. The measured oxygen production rate of 4-6 mmol/L culture h under continuous operation is consistent with the predicted performance of the unit for the provided light intensity.     

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.     

A fiber-optic retroreflective turbidimeter for continuously monitoring cell concentration during fermentation

A fiber-optic retroreflective turbidimeter has been developed to automatically and continuously assay the cell concentration in a fermentor by measuring the turbidity of the solution as a function of the light scattered at 180° to the incident light. The output signal is nearly directly proportional to the cell concentration in a fermentor when the sample stream contains from 0 to more than 50 g of cells per liter (wet weight). The device consists of a bifurcated fiber-optics light pipe with its distal end inserted into a flow cell through which the material to be analyzed passes. A light source on one proximal branch of the light pipe illuminates the sample stream; light that is back-scattered from participates in the stream re-enters the light pipe and is returned to a photodetector on the other proximal branch of the light pipe. A signal conditioning system connected to the optical head by a cable provides gain and zero adjustment.     

The use of flow cytometry to measure X-ray survival in cultured T lymphocytes

Light-scattering signals produced in a flow cytometer containing unstained, irradiated T-lymphocytes (MOLT-4 cell line) were analysed by plotting the axial light loss versus right-angle scatter. The resulting three-dimensional scattergram separated into two regions, corresponding to live and dead cells, as confirmed by trypan blue staining. The method is simple, rapid, allows large numbers of cells to be measured, avoids staining artifacts and is suitable for measuring radiation-induced killing down to 0.5 to 0.1 Gy.     

High-resolution 3-D imaging of living cells in suspension using confocal axial tomography

Conventional flow cytometry (FC) methods report optical signals integrated from individual cells at throughput rates as high as thousands of cells per second. This is further combined with the powerful utility to subsequently sort and/or recover the cells of interest. However, these methods cannot extract spatial information. This limitation has prompted efforts by some commercial manufacturers to produce state-of-the-art commercial flow cytometry systems allowing fluorescence images to be recorded by an imaging detector. Nonetheless, there remains an immediate and growing need for technologies facilitating spatial analysis of fluorescent signals from cells maintained in flow suspension. Here, we report a novel methodological approach to this problem that combines micro-fluidic flow, and microelectrode dielectric-field control to manipulate, immobilize and image individual cells in suspension. The method also offers unique possibilities for imaging studies on cells in suspension. In particular, we report the system's immediate utility for confocal "axial tomography" using micro-rotation imaging and show that it greatly enhances 3-D optical resolution compared with conventional light reconstruction (deconvolution) image data treatment. That the method we present here is relatively rapid and lends itself to full automation suggests its eventual utility for 3-D imaging cytometry.     

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.     

EMOSS: an epiillumination microscope objective slit-scan flow system

An epiillumination microscope objective slit-scan flow system has been fabricated utilizing two dimensional slit scanning with hydrodynamic sample stream focussing. Low resolution (4 micron) analysis of cellular fluorescence is facilitated by the definition of a stabilized flow plane through hydrodynamic focussing. Coincidence of the region of stabilized flow with the focal plane of the microscope objective will allow for the collection and subsequent imaging of fluorescence from cells oriented along this plane. Two orthogonal slit-scan contours are generated as a cell traverses the excitation region. It is hoped that the need for a three dimensional system will be precluded by preferential orientation of the cells in the region of stabilized flow. Cellular fluorescence is collected by a high numerical aperture epiillumination optical system and imaged onto two orthogonal slits. Two photomultiplier tubes are used to detect fluorescence. It is anticipated that the epiillumination microscope objective slit-scan flow system will be used with a variety of fluorescent stains and markers, as well as extended to the research of light scattered by cells. (Steen, H.B., Cytometry 1:26-31, 1980.     

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.     

FLOW CYTOMETRY AND THE SINGLE CELL IN PHYCOLOGY

Flow cytometers measure light scattering and fluorescence characteristics from individual particles in a fluid stream as they cross one or more light beams at rates of up to thousands of events per second. Flow cytometrically detectable optical signals may arise naturally from algae, reflecting cell size, structure, and endogenous pigmentation, or may be generated by fluorescent stains that report the presence of otherwise undetected cellular constituents. Some flow cytometers can physically sort particles with desired optical characteristics out of the flow stream and collect them for subsequent culture or other analyses. The statistically rigorous, cell‐level perspective provided by flow cytometry has been advantageous in experimental investigations of phycological problems, such as the regulation of cell cycle progression. The capacity of flow cytometry to measure large numbers of cells in large numbers of samples rapidly and quantitatively has been used extensively by biological oceanographers to define the distributions and dynamics of marine picophytoplankton. Recent work has shown that flow cytometry can be used to elucidate relationships between the optical properties of individual cells and the bulk optical properties of the water they live in, and thereby may provide an explicit link between algal physiology and global biogeochemistry. Unfortunately, commercially available flow cytometers that are optimized for biomedical applications have a limited capacity to analyze larger phytoplankton. To circumvent these limitations, many investigators are developing flow cytometers specifically designed for analyzing the broad range of sizes, shapes, and pigments found among algae. These new instruments can perform some novel measurements, including simple fluorescence excitation spectra, detailed angular scattering measurements, and in‐flow digital imaging. The growing accessibility and power of flow cytometers may allow the technology to be applied to a wider array of problems in phycology, including investigations of nonplanktonic and multicellular algae, but also presents new challenges for effectively analyzing the large quantity of multiparameter data produced. Ultimately, the detection of molecular probes by flow cytometry may allow single‐cell taxonomic and physiological information to be garnered for a variety of algae, both in culture and in nature.     

Photobiological and thermal effects of photoactivating UVA light doses on cell cultures.

While near-ultraviolet light has been widely used to photoactivate fluorophores and caged compounds in cells, little is known of the long-term biological effects of this light. UVA (315-400 nm) photoactivating light has been well characterized in short-term cell studies and is now being employed in higher doses to control longer-duration phenomena (e.g. gene expression). Annexin V-Cy5/propidium iodide apoptosis flow cytometry assays were used to determine responses of HeLa cells to doses of UVA light up to 23.85 J cm(-2). Cells seeded at low densities had higher percentages of apoptosis and necrosis and were also more susceptible to UVA damage than cells seeded at higher densities. The dose to induce apoptosis and death in 50% of the cells (dose(1/2)) was determined for two different commercially available UVA light sources: 7.6 J cm(-2) for the GreenSpot photocuring system and 2.52 J cm(-2) for the BlakRay lamp. All BlakRay doses tested had significant cellular responses, whereas no significant cellular responses were found for doses below 1.6 J cm(-2) from the GreenSpot light source. A temperature control and measurement system was used to determine direct heating from the UVA sources and also the effect that cooling cell cultures during photoexposure has on minimizing cell damage. Cooling during the BlakRay photoexposure significantly reduced the percentage of necrotic cells, but there was no significant difference for cooling during photoactivation with the GreenSpot. Differences in cell responses to similar UVA doses of different intensities suggest that photoduration should be considered along with total dose and thermal conditions in photoactivation studies.     

Isolation of viable type II alveolar epithelial cells by flow cytometry

We developed a new method for isolating viable type II cells from fractionated and unfractionated lung cell suspensions by flow cytometry using acridine orange (AO). Fischer-344 rat lungs were dispersed into single-cell suspensions by a technique that yields a high number of cells (4-5 X 10(8) cells/lung, congruent to 85% viable), congruent to 11% of which are type II cells. Elutriated fractions from the lung cell preparation and parent, unfractionated cell suspensions were incubated with 1.0-0.02 micrograms/ml AO and analyzed by flow cytometry. Parameters analyzed included axial light loss (ALL) and red fluorescence (RF). Based on their unique RF, attributable to AO staining of type II cell lamellar bodies, and their ALL characteristics, type II pneumocytes were sorted from elutriated fractions to greater than 95% purity. Using the same approach, type II pneumocytes were sorted from unfractionated lung cell suspensions at greater than or equal to 85% purity. The viabilities of the type II alveolar epithelial cells isolated by this method range from 85% to 95%, and the ultrastructural features of the sorted cells were unaltered by AO labeling or sorting.     

Selective electrofusion of conjugated cells in flow.

Using a modified flow cytometer we have induced electrofusion of K562 and L1210 cells in flow. The two cell types are stained with two different fluorescent membrane probes, DiO and DiI, to facilitate optical recognition, and then coupled through an avidin-biotin bridge. In the flow cytometer, the hydrodynamically focused cells and cell pairs are first optically analyzed in a normal flow channel and then forced to flow through a Coulter orifice. If the optical analysis indicates that a cell pair is present, an electric pulse is applied across the orifice to induce fusion. The pulsed cell pairs were subsequently analyzed using normal and confocal microscopy to evaluate fusion induction. It appears that fusion can be induced in about 10% of pulsed cell pairs when one electric pulse with a duration of 10-15 microseconds and an effective electric field strength of 4-8 10(5) V/m is used.     

Quantification of photonic localization properties of targeted nuclear mass density variations: Application in cancer‐stage detection

Light localization is a phenomenon which arises due to the interference effects of light waves inside a disordered optical medium. Quantification of degree light localization in optical media is widely used for characterizing degree of structural disorder in that media. Recently, this light localization approach was extended to analyze structural changes in biological cell like heterogeneous optical media, with potential application in cancer diagnostics. Confocal fluorescence microscopy was used to construct “optical lattices,” which represents 2‐dimensional refractive index map corresponding to the spatial mass density distribution of a selected molecule inside the cell. The structural disorder properties of the selected molecules were evaluated numerically using light localization strength in these optical lattices, in a single parameter called “disorder strength.” The method showed a promising potential in differentiating cancerous and non‐cancerous cells. In this paper, we show that by quantifying submicron scale disorder strength in the nuclear DNA mass density distribution, a wide range of control and cancerous breast and prostate cells at different hierarchy levels of tumorigenicity were correctly distinguished. We also discuss how this photonic technique can be used in examining tumorigenicity level in unknown prostate cancer cells, and potential to generalize the method to other cancer cells.