An analysing flow cytometer, the optical plankton analyser (OPA), is presented. The instrument is designed for phytoplankton anaysis, having a sensitivity comparable with commercially available flow cytometers, but a significantly extended particle size range. Particles of 500 pm in width and over 1,000 pm in length can be analysed. Sample flow rates of up to 55 pl/s can be used. Also, the dynamic range of the instrument is significantly increased for particles larger than about 5 pm. The optics, hydraulics, and electronics of the instrument are described, including the best form for a low fluid shear cuvette. The new pulse quantification technique we call digital integration is presented. This technique is essential for the instrument to handle both short and very long particles with a large dynamic range. Test measurements demonstrating particle size range and dynamic range are presented. Dynamic ranges of 10,000 and 100,000 were typically observed, measuring field samples with Microcystis aerug i n o s a colonies, whereas one sample showed a dynamic range of lo6. A simple method for interpretation of time of flight (TOF) data in terms of particle morphology is presented. The specifications of the instrument are given.

In flow cytometry, the coincident arrival of particles becomes a major problem when high sample rates are required. For the development of our high-speed photodamage flow cytometer (ZAPPER), it was of importance to understand the behavior of cells at flow rates of around 50,000-250,000 event/s. We developed and compared two models that describe the relation between the real cell rate and the detectable single cell rate. Both the Computer Simulation model and the Input/ Output Device model show distinct optima for the cell rate. The models were compared to measurements performed on the ZAPPER-prototype. Fits of the two models to the experimental data were excellent for cycle times of 4 and 15 p s and acceptable for a 2 p s cycle time. A third model (Mercer WB, Rev. Sci. Instr. 37:1515-1521,1966) could be fitted to the experimental data, after the proportionality constant k was adapted to the experimental data. At a yield of detectable single cells of 70%, the maximum cell rates are 180,000, 100,000, and 40,000 cells/s for cycle times of 2, 4, and 15 ps, respectively. Based on these results we can now select an optimal cell rate for analysis and sorting based on criteria such as accepted cell loss. In addition, the advantages of reducing the cycle time can now be evaluated with respect to the costs of that modification.Key terms: Cell sorting, poisson, simulation, ZAPPER Early cell sorters based on the fluid switching design (2,5) sorted a single cell in about 3 ms. Droplet sorters (3) greatly improved the sorting speed. Typically, the time needed to deflect a single cell in a single droplet is in the order of 30 ps. When three droplets per cell are deflected, a FACS I1 sorter checks 2n-1= 5 droplets for coincidence (12). This increases the time in which coincidence could occur from 30 to 150 ps, thereby reducing the maximum cell rate. Sorting two or three droplets per cell is common practice as it increases the chance that the desired cell is deflected. When high sort rates are important, sorting one droplet per cell is advisable (1). Considering that the electronics, such as amplifiers and analog to digital converters (ADCs) can handle pulses of 4 ps, droplet sorting is clearly limited by the droplet frequency. Higher frequencies require smaller nozzle orifices or increased sheath fluid pressure. Nozzles smaller than 50 pm are undesirable because of clogging.High-pressure flow cytometers, such as the Lawrence Livermore National Laboratory (LLNL) high-speed sorter (91, operate at a sheath fluid pressure of 14 atm (jet speed 50 mis), a cell rate of 22,OOOis, and a droplet frequency of 220,OOOis. At these settings one out every ten droplets is occupied by a n event. However, the viability of the cells sorted at that pressure varied. Chinese hamster ovary cells survived (83%) passage through the nozzle but a muriine bone marrow sample failed to develop any colonies upon subsequent culture.Photodamage cell selection (7,101 is a relatively new approach to cell sorting. The ZAPPER (4) is a flow cytometer equipped with ...

A flow cytometer was developed for the highspeed "sorting" of desired cells by selectively irradiating (zapping) the undesired cells from a population. After previous efforts to photoinactivate cells with photosensitizers had failed, it was decided to exploit the photosensitivity of the cell's DNA at 257 nm. It was shown that a 257 nm laser output power of 20-100 m W was s d c i e n t to induce a 4.5 log cell kill after the cells were processed through a focused 257 nm laser beam. Experiments proved that the photodamage flow cytometer (ZAPPER) could selectively photoinactivate cells at rates over 22,000 events/s, and selection purities ranged from 81% to 100%.The yields of the desired cells depended on the selection mode. In the Enrichment mode, the zap laser was not aimed at the jet, and only undesired cells were exposed to a brief ultraviolet (w) pulse after modulation of the UV laser beam. The yields of desired cells ranged from 95% to 105%. In the Purge mode, the zap laser beam was aimed onto the jet, and only desired cells were allowed to pass after deflection of the UV laser beam; the yields of desired cells ranged from 12% to 52%. The cause of the reduced yields in the PURGE mode was traced to the Eact that the Electro-Optic Modulator was used to modulate the zap laser proved too slow for the intended application.The lifetime of the frequency-doubling crystal used for the generation of the 257 nm beam was found to be limited to several days. These technical limitations could theoretically be overcome by the application of large argon ion lasers capable of emissions at 257-275 nm and an Acousto-Optic Modulator to deflect the zap laser. Key terms: AOM, EOM, flow cytometry, instrumentation, sorting, W, ZAPPER Selective photoinactivation of cells in a flow cytometer ( 3 , 9 ) could be a high-speed alternative to droplet sorting for the depletion of an undesired cell population, such as cancer cells (14), from a cell sample. Photoinactivation is generally accomplished by irradiating with a lethal fluence of light cells that have taken up a photosensitizer. Initial data ( 3 ) showed that the photoinactivation of cells that were incubated with 5-bromo-2'-deoxyuridine (BrdUrd) and Hoechst 33342 was extremely effective after a brief (5 ps) irradiation in a modified droplet sorter. However, the BrdUrd'Hoechst 33342 photosensitizer was abandoned, because its application was limited to actively cycling cells, which required culturing the cells with the photosensitizer for 2-3 days and proved impractical due to the extreme photosensitivity of the cells. Despite efforts to replace the BrdUrd'Hoechst 33342 photosensitizer, no effective alternatives were found. Finally, it was decided to exploit the intrinsic photosensitivity of cellular DNA.This approach presented several advantages. First, low fluences (<50 J/m') of short-wavelength ultraviolet light ( W C ) had been shown to photoinactivate cells effectively (1,4,8,16). Cell death was attributed to the two prevalent DNA photoproducts, i.e., the pyrimidine dimer and th...

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A new data acquisition and analysis format (classmode) was developed that allows real-time data classification in a flow cytometer. In our cytometer, detected events were classified in real time by their presence or absence in a set of look-up tables (LUT). A modification of the cytometer hardware allows the exclusive transfer of the LUT data to the acquisitiodstorage computer. Using a combination of 8 LUTs, the analyzed events can be classified into 256 subpopulations. Real-time data classiflcation results in an increased data transfer rate and a significant compression of the data. Q 1995 WiIey-Liss, Inc.