Anal. Chem. 1995, 67, 2666-2671
An All-Solid-state Flow Cytometer for Counting Fluorescent Microspheres Stefan Niehren* and Wolfgang Kinzelbach Institute of Environmental Physics, University of Heidelberg, Im Neuenheimer Feld 366, 69 120 Heidelberg, Germany
Stefan Seeger and JUrgen W o l f ” Institute of Physical Chemistty, University of Heidelberg, Im Neuenheimer Feld 253, 69 120 Heidelberg, Germany
Particle tracers are of interest in the investigation of fractured media. Because their transport behavior Mers from that of solute tracers, they supply complementary information on fracture space. This infomation is crucial in the risk assessment of underground hazardous waste storage sites. An easy-to-handleparticle tracer consists of fluorescentpolystyrene spheres with diameters on the order of 1 pm. Up to now, detection and counting of these particles was performed by fluorescence microscopy of filtered samples. To allow faster and on-line continuous monitoring of single particles, a flow cytometer has been developed in the present work. The cytometer is built from solid-stateoptical components, resulting in a robust and transportable system suitable for field use. To maximize the fluorescencequantum efficiency,a new dye, JA22, was used. Use of an avalanche diode allowed the efficiency of photon detection to be maximized. For complete digital data acquisition and analysis, only a personal computer is required. The detection dciency of the cytometer is >25%,the probe flow is 20 mWh, and the present accuracy is 160 particles/mL. The development of new methods of research on flow and filtration of groundwater in aquifers has increased considerably in the last decade.’-4 The interest is motivated by groundwater pollution, remediation, and hazardous waste repository assessment. A common method of obtaining information on transport phenomena in aquifers is to observe natural or artijicial tracers carried along by groundwater flow. Most investigations use heat or solute tracers such as radioactive ions, fluorescent dyes, and salt. If information about the filtration mechanism in an aquifer or about the size distribution of fractures or holes is necessary, particles give additional and complementary information due to their spatial extent. Filtration is a decisive factor in self-puri6cation of groundwate+ for drinking purposes. A further field of research (1) de Marsily, G. Quantitative Hydrogeology; Academic Press, Inc.: Orlando, FL, 1986. (2) Matthess, G.; Ubell, K. Allgemeine Hydrogeologie Grundwasserhaushalt; Gebriider Borntaeger: Berlin, Stuttgart, 1983. (3) Kobus, H. E.; Kinzelbach. W. Contaminant Transport in Groundwater, A. A. Balkema: Rotterdam, Brookfield, 1989. (4) Dracos, T. H.: Stauffer, F. Transport and Reactive Processes in Aqulfen; A. A Balkema: Rotterdam, Brookfield, 1994. ( 5 ) Yao, K. M.; Habibian, M. T.; O’Melia. C. R Environ. Sci. Technol. 1971,5, 1105-1112.
2666 Analytical Chemistry, Vol. 67, No. 75,August 7, 7995
is the storage of hazardous waste. Safety analysis mostly considers only the transport of harmful substances dissolved in the fluid phase, but less mobile substances can also be transported due to their sorption on mobile colloids!-* Colloids may move faster than solutes due to preferential transport in larger fractures and the lack of retardation by matrix diffusion. The third field of interest is the transport of bacteria in a heterogeneous medium, which arises in the transmission of some waterborne diseases? in biorestoration of organically contaminated aquifers using nonindigenous or waste-adapted populations of microorganisms,1° and in microbially enhanced oil recovery.l* The result of a tracer experiment is a curve of tracer concentration versus time, a so-called breakthrough curve. Its particular shape contains information on the distribution of travel times between the injection and the measuring points. Reliable and automatic tracer detection is crucial for the performance of a long-term continuously monitored tracer experiment. While continuous monitoring poses no problem in experiments with solute tracers, it is still not available for particle tracers. Commonly used particle tracers are bacteria or fluorescencelabeled polystyrene spheres. Bacteria can be detected either by cultivation of bacteria populations and counting of these cultures or by labeling bacteria with fluorescent dyes like DAFT1* Fluorescence-labeled particles or bacteria can be detected with a fluorescence microscope after filtration of the water sample with a fine filter and counting of the fluorescent particles. This procedure takes a long time and is therefore not suited to on-line detection or continuous measuring. The motivation of our investigation is to construct a small, robust detector suited for in situ and on-line detection. Flow cytometry, common in biology and medicine, can provide a solution, given the accuracy and versatility of this technique. Flow cytometry instruments are normally used to analyze and classify biological particles such as immune complexes, individual (6) McDowell-Boyer, L. M.; Hunt, R. J.; Sitar, N. Water Resour. Res. 1986,22,
1901-1921. (7) Buddemeier, T. W.; Hunt, J. R. Appl. Geochem. 1988,3,535-548. (8) McCaulou, D. R.; Bales, R. C.; McCarthy. J. F. J. Contam. Hydrol. 1993, 13, 167-181. (9) Bitton, G.; Harvey, R. W. New Concepts in Environmental Microbiology: Wiley-Liss: New York, 1992; pp 103-124. (10) Wilson, J. T.; Leach, L. E.; Henson. M.; Jones, J. N. Ground Water M o n k Rev. 1986,6, 56-64. (11) McLeod, F. A; Lappin-Scott, H. M.; Costerton, J. W. Appl. Environ. Microbiol. 1988,54, 1365-1372. (12) Harvey, R. W. Water. Resour. Res. 1993,29, 2713-2721. 0003-270019510367-2666$9.0010 0 1995 American Chemical Society
virus particles, liposomes, cellular organelles, bacteria and fungi, chromosomes. cells, or cell aggregates. These particles flow through a sensing region in which electrical resistance or optical properties are measured. The most sensitive system uses the detection of fluorescent light emitted by particles labeled with a fluorescent dye. Normally, the dye will be excited by a laser system with an exactly defined wavelength. The emitted light is collected by a microscope object lens and detected by a photomultiplier. Most of the available fluorescent dyes are blue, green. or Wexcitable, so argon ion, krypton ion. neodymYAG, or nitrogen lasers are used for the excitation. These lasers are expensive and bulky, and their lifetime is limited. The smallest and cheapest laser systems available are laser diodes. Up to now. such diodes emitted light from near-infrared (1950 nm) to red (629 nm) at room temperature. The power of these lasers depends on their wavelength and reaches 15 mW at 629 nm. We chose a single mode laser diode with an output power of 8 mW at a intrinsic wavelength of 629 nm. With a low-power laser, it is necessary to detect as much emission light as possible. The detector with the best efficiency is an avalanche photodiode, which reaches 80%for red and nearinfrared light. The avalanche photodiode used is a single photon counting avalanche diode (SPAD). Technical details are given in ref 13. For the above reasons, it is necessary to use optical solid state components to build a small, robust, and easily transportable system.".'" Additionally, we are interested in the red light excitation in order to maximize the selectivity of the detector. Organic molecules show fluorescence in the green. blue, or ultraviolet frequency range. so organic colloids in the groundwater and labeled particles are distinguishable by using a red fluorescent dye. We chose the dye JAZZ because of its quantum efficiency of 89.94;.'" Detailed descriptions are given in the next section.
-
.-x v
c aJ
.-c aJ .->
wavelength in nm Figure 1. Emission and absorption curves of the fluorescence dye JAZZ." Additionally. the chemical structure is given. mirror
hicovex tense
v
excitation filter
I!!
aspheric
lenses
-
EXPERIMENTAL SECTION Particle Preparation. The particles we employed were
polystyrene microspheres F'olyscience Ltd.. Eppelheim, Germany) with a diameter of 1 pm. The diameter was determined with a standard deviation of 3%. To label the particles, we put the aqueous particle suspension (2.5% solids latex) in a dialysis bag'* and equilibrated it over a period of 8 days with an external aqueous solution saturated with the organic fluorescent dye JAZZ. The suspension was then equilibrated for another 8 days with pure water so that the concentration of the dye was minimized in the aqueous part of the particle suspension. We observed aggregation of several particles in a microscope. M e r 15 min. ultrasound treatment disaggregation occurred. The dye JAZZ was designed for fluorescence spectroscopy in hioanalytic applications and has a quantum efficiency of 89.9?iI6 (13) Li-QuanK. I-; Iloyd. \1. I). Reo. Sei. Instmm. 1993. 64, 1524-1529, (14) Doombor. R. M. I,.;DrGrooth. H. (;.; Kraan. Y. M.:Van Der P o d C. J.: (;YPY(., J. C y f o m r f y1994. 15. 267-271. (151 Doornbos. R. M. I,.:Hcnnink. E. J.: Putman. C. A J.: De Grooth. B. C.: Grrvr~.J. Cyfomefv 1993. 14. 5R9-594. Ufi) Caurr. M.:Nan. K-T.;%hull A:'Tadd;>y,R !%p~ger,S.:Wolfrum. J.: ArdmJacob. J.: I)rllau. (;.: Mam. H.: 1)rcxhaKc.. K I I . I Fltiorssr. 1993.3.(3) 131 - 13!1. 117) Ardmlarob. J. NWP IonRuelliRe XonthmFor6sfo.efi;r Fluorpurnmmden und Forbsfofloser Vrrlay Shaker: S i r ~ ~ ILT)Z. n. (1.81 Hsiao. J.-S.: Wrbhrr. S.E.J.Phys. Chrm. 1993. 97,XZR9-R295.
flow chamber
Figure 2. Basic layout of our instrument. The dye molecules are excited by a diode laser. Fluorescent light is detected orthogonally to the llow/excitalion plane. The emitted light is collected by two aspheric lenses and detected by a single photon counting avalanche diode (SPAD).
Its excitation maximum is at 621 nm, the emission maximum at 642 nm, its excited state lifetime is 4.06 ns. and the absorption cross section is 1.1 x l@L/(molrm)." These data were measured in ethanol; data in polystyrene are not available. The emission and absorption curves and the chemical structure ofJA22 are given in Figure 1. Instrumentation. Figure 2 shows the optical design of our instrument. The basic layout is similar to that of most single laser systems.Ig The fluorescent light is detected orthogonally to the flow/excitation plane. A significant difference from most single laser systems is that we do not detect forward scattered light. To excite dye molecules adsorbed on the microspheres, we used the diode laser APM08635 (laser Graphics, Diehurg. (19) Shapiro. H. M. PmctiralNou Cylomety: Alan R. Iiss. In
