FEMS Microbiology Reviews 32 (1986) 159-164 159 Published by Elsevier

FER 00010

Application of flow cytometry to algal physiology and phytoplankton ecology

(Aquatic particle analysis; water quality monitoring)

Alexander C u n n i n g h a m and John W. Left ley

Department of Applied Physics, Strathclyde University, 107 Rottenrow, Glasgow G40NG, and Scottish Marine Biological Association, Dunstaffnage Marine Research Laboratory, P.O. Box No. 3, Oban, PA34 4AD, U.K.

Received 22 August 1985 Accepted 19 September 1985

1. SUMMARY

Flow cytometry, originally developed for bio- medical purposes, is now being put to extensive use in the study of microalgae. Successful applica- tions to date include studies of cytochemistry, cell cycle dynamics and phytoplankton ecology. This review summarises recent work in a rapidly expan- ding field of research, and highlights those areas in which significant progress can be expected when the necessary equipment becomes more readily available.

2. INTRODUCTION

In flow cytometry, cells suspended in a stream of fluid are presented singly to a source of intense illumination. Optical signals such as light scatter- ing and fluorescence from individual cells are mea- sured and logged at a typical throughput rate of 1000 cells/s (Fig. 1). Some instruments can mea- sure cell volume electronically, using the Coulter principle. The more advanced machines have a sorting facility, in which the cell-bearing fluid stream is broken into droplets which can be de- flected electrostatically into one of three con-

tainers. Decisions on the direction of deflection are made by electronic circuitry on the basis of the optical signals received, using criteril| which can be adjusted by the operator. This allows sub-popula- tions with predetermined optical characteristics to be isolated for microscopic examination or further culturing. In principle, a mixture of particles could also be sorted for chemical analysis, but the very small mass of material that can be sorted in a reasonable period of time makes this practical only if very sensitive analytical methods can be em- ployed.

The development of flow cytometry as a tool suitable for routine use in the biological laboratory was carried out mainly during the 1970's [1] but it is only recently that the technique has been ap- plied to the study of algae. Commercial instru- ments are now manufactured by Coulter Electron- ics, Becton Dickinson, and Ortho Diagnostics: the current state of development of research instru- ments is reviewed by Steinkamp [2]. A major new textbook on the technique has just been published [3]. Instruments vary in the type of illumination used (mercury arc lamps or single or multiple lasers), the presence or absence of a sorting facil- ity, and the degree of sophistication of the data handling. The most modern machines are micro-

0168-6445/86/$03.50 © 1986 Federation of European Microbiological Societies

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Fig. 1. Schematic diagram of a flow cytometer. Detector ] collects forward scattered light, detector 2 collects right-angle scatter and detectors 3 and 4 collect fluoresocnce at two different wavelengths. The fluid stream is broken into droplets which are electrically charged before they pass through the deflection plates. For the analysis of microalgae by autofluores- cence, the illuminating beam would typically have a wavelength of 450-500 nm, with fluorescence measurements being made at 550-600 nm (phycoerythrin) and > 650 nm (chlorophyll).

processor-controlled and can be operated from a keyboard: experimental protocols, once developed, can be stored on disc for routine use. While the standard of design and construction of commercial flow cytometers is high, these instruments are very costly in comparison with the usual tools of the phycological laboratory. Consequently, several workers have constructed simplified flow cytome- ters for their own use [4,5]: the most successful of these have been built around fluorescence micro- scopes [6-8].

Recently, there has been a growing realisation of the potential power of flow cytometry as a tool

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LRF Fig. 2. Analysis of marine microalgae by flow cytometry. (a) It is possible to discriminate between two species of ultraplankton with cells of similar diameter, using 2-wavelength fluorescence measurements. Peak 1 is Synechococcus sp. DC2 (Cyanophy- cae, approx. 2 ttm); and peak 2 is 12 48-23 (Prasinophyceae, approx. 3 am). (b) Discrimination of cells with similar fluores- cence characteristics using forward light scatter as an ad- ditional parameter. Peak 1 is DC2 (approx. 2 ttm); peak 2 Pavlova lutheri (approx. 5 #m). Algae were obtained from the Scottish Marine Biological Association Culture Collection. Axis labels are: N, number of events; LOF, log orange fluorescence; LRF, log red fluorescence; LFS, log forward light scatter.

for phytoplankton research [9], and full-scale com- mercial instruments are now being used in two laboratories in the United States (Woods Hole and the Bigelow Laboratory for Ocean Sciences). One will shortly be in use in Europe (at the Institute for Marine Environmental Research, Plymouth, En- gland), with the acquisition of more machines by laboratories in various countries being likely in the immediate future. Fig. 2 shows examples of the

analysis of mixtures of cultured marine algae car- ried out on one commercial machine, the Coulter 'EPICS C'. However, the most widespread current use of flow cytometers is still in biomedical labora- tories, in areas such as blood cell analysis, tumour screening and flow karyotyping. The adaptation of existing experimental methods, which have been developed using mammalian cells, to the study of microalgae is not without difficulties. This review therefore has two purposes: firstly, to summarise the existing work on algae which forms the foun- dation of a rapidly growing field of research; and secondly, to highlight those areas in which the application of flow cytometry to the study of microalgae offers most promise of progress in the immediate future.

3. METHODS

3.1. Induced fli~orescence

Most of the staining techniques developed for fluorescence microscopy can be used in flow cy- tometry. Using such methods, it is possible to use fluorescence signals to quantitatively measure DNA, RNA, and total protein per cell, to estimate intracellular pH and membrane potential, and to test for cell viability [2,10]. In addition, cell surface markers can be labelled using fluorochrome-con- jugated antibodies, opening up the possibility of rapidly distinguishing between closely related organisms such as chroocxx:c, oid cyanobacteria [11]. By a careful choice of fluorochrome absorption and emission spectra, the presence of up to three cell labels can be discerned simultaneously using a single illuminating wavelength (usually the 488 nm line from an argon-ion laser). The availability of these staining procedures transforms the flow cy- tometer into an effective tool for histochemical assays. Virtually all the measurements could be made using a fluorescence microscope equipped with a suitable photometer, but flow cytometry allows large numbers of cells to be analysed for several properties in a very short time. This makes it a simple matter to determine the distribution of properties within a population. In particular, since cells can be assigned to the G1, S, or G2 phases of

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the cell cycle using DNA stains, it is possible to use additional stains simultaneously in order to monitor the phase relationship between DNA synthesis and other aspects of the cells' metabolic activity.

The prospect of applying such powerful tech- niques to problems in algal physiology is clearly an exciting one, but the transfer of the methodol- ogy from mammalian to plant cells does pose some problems. The main features of algal cells that give rise to difficulty in this respect are the presence of cell walls which hinder stain penetra- tion, of cytoplasmic structures which retain stains destined for the nucleus, and of absorbing pig- ments which may interfere with fluorescence [12].

In spite of these difficulties, modified protocols for staining algae have been successfully developed [8,12-14]: in many cases, the most important ad- ditional step is the bleaching of the cells to prevent interference by pigments [8,9,13,14]. This is usu- ally done by solvent extraction, but photo-oxida- tion has been used successfully in for the dinofla- gellate Gonyaulax tamarensis [15]. Work on the cytometric assay of the saxitoxin content of this species by measuring signals from a fluorescent derivative of the toxin provides an example of an elegant alternative to the use of fluorochromes in this field [16]. It also provides an indication that as work progresses, we can expect to see cytochem- ical procedures evolve which exploit the unique physiological characteristics of algal cells.

3.2. Autofluorescence

While the presence of photosynthetic pigments complicates staining procedures, it does lead to greatly enhanced opportunities for the study of live cells using autofluorescence. The complexity of the multi-pigment light-harvesting systems in microalgae means that intact cells have broad-band excitation and emission spectra [17,12]. However, most flow cytometers are limited to single-band illumination, and 2- or 3-band fluorescence detec- tion, and so it is necessary to adopt a rather simple scheme of fluorescence analysis. The usual strategy is to illuminate the cells with light in the wave- length region of 450-500 nm (the 488 nm argon line is commonly used) and to use band-pass filters

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to collect fluorescence at 550-600 nm (characteris- tic of phycoerythrin) and 650-700 nm (character- istic of chlorophyll). The intensity of the fluores- cence signal is clearly related to the quantity of pigment contained in the cell [13] but the relation- ship is not one of absolute proportionality, since the physiological state of the cells and their previ- ous illumination history have to be taken into account [14,18]. Nevertheless, even relative mea- surements of pigment fluorescence are extremely useful: firstly, for discriminating between algae and detritus and secondly, for discriminating be- tween algal types. For example it is possible to differentiate between Chlorella and Euglena cells grown under similar conditions simply on the ba- sis of an order of magnitude difference in the level of fluorescence per cell [13]. Moreover, since only cyanobacter ia and Cryp tomonads contain phycoerythrin, the presence of cellular fluores- cence in the 550-600 nm waveband is diagnostic for the presence of these groups [17,18]. Chloro- phyll fluorescence disappears in cells poisoned by heavy metals, and flow cytometry can be used to follow the kinetics of onset of the intoxication process [19].

3.3. Light scattering

Most flow cytometers measure the light scattered by cells into two solid angles, oriented along the direction of propagation of the il- luminating beam and at right angles to it. Exact optical calculations of scattering intensities are not possible for particles with the intricate structure of algal cells, but it can be briefly stated that the forward scattering signal carries information on cell size and shape, while the right-angle signal carries information on internal structure. In medi- cal cytometry, for example, a 2-dimensional plot of forward and 90 ° light scatter is widely used for discriminating between the various groups of white blood cells. Commercial cytometers currently mea- sure the total amount of light scattered in the forward direction, but the angular distribution of this light carries a great deal of information: the use of multi-angle scattering measurements for analysing bone marrow cells was suggested by Loken et al. [20]. The variety of sizes and shapes

found in microalgae is much greater than in mam- malian cells, and the potential for using scattered light as a discriminatory tool is consequently in- creased. For example, it is possible to distinguish between Chlorella, Chlamydomonas and Anacystis cells by measuring the angular distribution of the forward scattered light using a detector consisting of 32 concentric photosensitive rings [21]. Coher- ent optical image processing has been applied to microphotographs of diatom cells [22,23], and this approach is being extended at the Commission of the European Communities Joint Research Centre at Ispra, where Forrest and Rossi are applying pattern recognition techniques to the analysis of Fraunhofer diffraction patterns generated by lake phytoplankton in a flow cytometer [24].

4. APPLICATIONS

Flow cytometry has recently been used to study nuclear synchrony in the macroalga Valonia mac- rophysa [25]. However, most of the current interest in the application of the technique to phycology centres around the possibility of obtaining infor- mation on algal ecology that would otherwise be inaccessible. Samples of natural phytoplankton populations usually contain a wide range of other particulate matter such as microzooplankton, bacteria and detritus: the selective fractionation of this mixture is a perennial problem [26]. However, of all the components of a natural plankton com- munity only the algae show autofluorescence. They can therefore be readily identified, separated and further characterised with the aid of flow cytome- try. This ability to discriminate not only between algae and other microorganisms, but also between different classes of algae [9,12,18] opens up excit- ing possibilities for the ecologist. Following the initial published trials of Yentsch et al. [9], several laboratories have used flow cytometers for natural sample analysis, and one Coulter EPICS instru- ment has been taken to sea [27]. Successful appli- cations to date include: (1) The discrimination of algae from detritus using autofluorescence [9]. (2) The detection of small cyanobacteria in uncon- centrated samples and the isolation of viable cells for cloning [9].

(3) The study of variations in the size, number density and pigment content of a Synechococcus population with depth in the Gulf Stream [27]. (4) The investigation of the degree of selectivity in phytoplankton ingestion and digestion exhibited by the mussel Mytilus edulis [28].

In all the above applications, flow cytometry was used in close conjunction with fluorescence microscopy, the usual procedure being to use the cytometer to identify prominent features of the population in a suitably defined parameter space, and to inspect sorted cells under the microscope to enable taxonomic interpretations to be made. However, as multi-parameter 'fingerprints' are built up for commonly occurring organisms, it is possible that the taxonomic analysis of natural samples could be automated to some extent. One development that has been long heralded [12,19] is the application of flow cytometry to automatic water quality monitoring. This idea relies on the assumption th/~t the phytoplankton characteristic of water bodies in various stages of eutrophication may show distinct cytometric signatures; clearly, much work has still to be done in this area.

In order to exploit fully the ability of the flow cytometer to provide information on mixed popu- lations of cells, it is necessary to measure simulta- neously as many parameters as possible. However, the collection of 5 parameters per cell at a throughput rate of 1000 cells/s leads to the accu- mulation of very large quantities of data, and poses problems for subsequent data inspection and manipulation. It is probable that the next major developments in flow cytometry will be in the area of computer software rather than optics or flow system design.

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range of uses as existing multi-purpose instru- ments such as the fluorescence microscope, ultra- centrifuge, or Coulter counter. However, existing instruments are designed for biomedical use, and are not optimally configured for phycological re- search. Improvements could be made in the resolu- tion of light scattering measurements, in software for handling multi-parameter data sets and in the portability of the equipment.

As more instruments become available, it is likely that they will be widely used in laboratory studies in areas such as the growth of cultures under l ight/dark cycles, grazing experiments, multi-species competition, transient growth dy- namics, the variation of cell pigment content with light intensity, culture purification and the iso- lation of cells for cloning. On the ecological front, the rapid rate of sample analysis should make it possible to obtain profiles of the spatial and tem- poral variation of natural populations at a resolu- tion that was hitherto unobtainable. However, the current growth of interest and activity in the field makes this list of suggestions inevitably incom- plete. Perhaps the only safe prediction is that flow cytometry will have a major impact on the devel- opment of the study of algal physiology and ecol- ogy in the years ahead.

ACKNOWLEDGEMENTS

We thank Dr. J.I.M. Forrest for many informa- tive discussions on flow cytometry, and Coulter Electronics for the temporary use of a flow cy- tometer.

5. DISCUSSION

Flow cytometers are now established as a powerful addition to the range of instruments available to the student of algal physiology and ecology. The current range of application of the devices is limited only by their high cost, and by the time required to develop appropriate sample preparation techniques and experimental proto- cols. Potentially, flow cytometry has as wide a

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