Three-Color Fluorescence Measurements on Single Cells Excited at Three Laser Wavelengths

Transcription

1 /82/ $00.00/0 CYTOMETRY Copyright 0 by the Society for Analytical Cytology Vol. 2, No. 4, 1982 Printed in U.S.A. Three-Color Fluorescence Measurements on Single Cells Excited at Three Laser Wavelengths John A. Steinkamp, Carleton C. Stewart and Harry A. Crissman Los Alamos National Laboratory, Los Alamos, New Mexico Received for publication July 28,1981; accepted September 23, 1981 A three-laser flow cytometer for quantitative analysis and sorting of cells and microscopic particles has been developed and evaluated using argon- and krypton-ion lasers as excitation sources. Cells stained with three fluorescent dyes having different excitation spectra enter a flow chamber where they first pass through an electronic volume sensor and then intersect three spatially separated laser beams for fluorescence excitation of bound dyes and light scatter measurements. Separate pairs of beam-shaping optics independently position and focus each beam onto the cell stream thus permitting cells to be sequentially illuminated. Fluores- cence is measured by a detector that employs a single collecting lens for single or multilaser excitation experiments. Fluorescence signals are processed and displayed as frequency distribution histograms using an LSI-11 computer. This instrument is described in detail with illustrative examples using cells stained for DNA content, protein, and cell surface antigens and cells that have phagocytized fluorescent microspheres. Key terms: Flow cytometry, fluorescence, lasers, DNA content, protein, phagocytosis, cell-surface antigens The primary laser excitation wavelength used in flow cyto- lengths with spatially separated beams can be used to sequenmetric cell analysis and sorting systems (2,6-9,12, 17) is the tially excite different fluorochromes bound to a cell (1, 4, 11, 488 nm line obtained from argon-ion lasers. A number of 14, 16). fluorescent dyes, including acridine orange, fluorescein isothi- Described below is a three laser beam instrument for mulocyanate (FITC), propidium iodide, ethidium bromide, rho- tiparameter measurement of stained cells and fluorescent damine 123, and acriflavine are excitable at this wavelength. particles having different excitation and emission spectra. However, many stains require excitation at shorter or longer This instrument also contains additional electronic volume wavelengths. For example, mithramycin, chromomycin A3, and light scatter sensor channels and it was a developmental fluorescamine, and brilliant sulfaflavine excite best in the 400- outgrowth of previously described single and dual laser cell 450 nm wavelength region. 4-6-Diamidino-2-phenylindole separators (12, 14). To permit versatility in fluorescence mea- (DAPI), Hoechst, and 4-acetamido-4 -isothiocyanate-stilbene- surements, one krypton and two argon laser beams are inde- 2,2 -disulphonic acid (SITS) excite only in the UV (near 350 nm). Other dyes have primary excitation in the green, yellow, and red regions. Experiments often require more than one fluorescent dye to measure biochemical and functional properties of cells. Since stains of different wavelength excitation spectra are available, two or more lasers operating at different wave- This work was performed at the Los Alamos National Laboratory, Los Alamos, New Mexico under sponsorship of the United States Department of Energy. Presented at the Analytical Cytology Conference VIII, Westworthby-the-Sea, New Hampshire, May 19-25, pendently focused onto the cell stream using separate pairs of beam-shaping optics. The three-color fluorescence sensor, which uses a single collecting lens, contains a dichroic fiiter and mirror arrangement for positioning the sensing regions of two of the photodetectors onto, above, or below the other one for single or sequential multilaser measurements. Three-color fluorescence signals are processed and displayed as frequency distribution histograms using an LSI-11 computer (Digital Equipment Corp., Maynard, Mass.). This versatile system can be used for single or multilaser induced fluorescence and other optical measurements at wavelengths ranging from the uv to IR and will have immediate application in immunology, oncology, and hematology for analyzing mixed cell populations.

2 MULTILASER FLOW CYTOMETRY 227 FIG. 1. Cutaway view of the flow chamber illustrating three-laser excitation with spatial positioning of the beams. Materials and Methods As illustrated in Figure 1, cells stained with fluorescent dyes and suspended in normal saline are analyzed sequentially (lo00 cells/sec) as they pass through the flow chamber. On entering the chamber, they first pass through an electronic volume sensing orifice' and then intersect a krypton and two argon laser beams of appropriate wavelength (Table I) spaced pm apart. Two of the lasers are each placed 10" off the normal perpendicular beam axis to the flow cell and the third in the normal position, all in approximately the same geometrical plane. The three beams, which have Gaussian power density profiles (TEMw mode), are focused into elliptical cross-sections at the cell stream-laser beam(s) intersection by separate sets of crossed cylindrical lenses. The two outside beams and the middle beam are focused by 30 and 2.2 cm and 30 and 5.1 cm lenses, respectively. Micromanipulator lens mounts allow each beam to be raised, lowered, and focused so that cells can be sequentially illuminated. Fluorescence emitted by stained cells intersecting three laser beams is measured with sensors capable of detecting light in one, two, or three wavelength regions as selected by various filter combinations (Fig. 2). An f/1.6 projection lens collects emitted fluorescence and 90" light scattered as cells sequentially intersect the three laser beams. Barrier filter No. 1 blocks light scattered by cells intersecting laser beam No. 1 but passes fluorescence above the filter cutoff wavelength. Fluorescence and light scattered by cells intersecting beams Nos. 2 and 3 (longer wavelengths than barrier filter No. 1 cutoffl are passed by barrier fiter No. 1. Dichroic color-separating filter No. 1 in * Not used for data presented in this paper. combination with barrier No. 1 and shortpass No. 1 fiiters determines the fluorescent channel No. 1 wavelength passband. This typically ranges from nm (blue-violet). A lens images (1:l) laser beam No. 1-cell stream intersection onto a 200 pm diameter pinhole located in front of photomultiplier tube (PMT) No. 1. Dichroic filter No. 1 also reflects fluorescence and light scattered (typically above 500 nm) to barrier filter No. 2 from cells intersecting beams No. 2 and 3. Barrier fdter No. 2 blocks light scattered by cells intersecting beam No. 2, but passes fluorescence above the filter cutoff wavelength. Fluwescence and light scattered by cells intersecting beam No. 3 are passed by barrier filter No. 2. Dichroic filter No. 2 reflects colored light (typically below nm) to shortpass filter No. 2. Therefore, the combination of barrier No. 2, the 45" reflecting characteristics of dichroic No. 2, and shortpass No. 2 filters determine the fluorescence channel No. 2 wavelength passband. This normally ranges from nm. Similarly, a second lens images the laser beam No. 2-cell stream intersection onto a 200 pm diameter pinhole in front of PMT No. 2. Light transmitted by dichroic filter No. 2 (above nm) is reflected by a 45' front surface mirror to barrier filter No. 3 which blocks light scattered by cells intersecting beam No. 3. Fluorescence excited by beam No. 3 is subsequently passed by the barrier filter and focused by a lens onto a 200 pm diameter pinhole in front of PMT No. 3. Depending upon barrier filter No. 3 and the spectral properties of the dichroic filters, the channel No. 3 fluorescence measurement region normally ranges from 600 nm to PMT No. 3 cutoff wavelength. The spatial location of pinhole Nos. 2 and 3 with respect to pinhole No. 1 can easily be viewed with a microscope when they are illuminated by small incandescent lamps placed in front of the PMT. This

3 228 STEINKAMP, STEWART AND CRISSMAN Table 1 Wavelengths and Powers Available with the CR-4 and CR-10 Argon and CR-3OOOK Krypton Lasers" Wavelength Power (watts) (nm) CR-4 CR-I0 CR-3000K Ultraviolet 333, 351(2), , 350,356 Violet Blue Green Yellow Red Infrared " Coherent, Palo Alto, California. I.o permits aligning of the cell stream-laser beam intersections onto the pinhole images (Fig. 2-flow chamber front view). If multilaser excitation is selected, laser beam No. 1 and the cell stream are made to intersect in the center of pinhole No. 1 image (blue). Dichroic filter No. 2 is then positioned by micrometer adjustment to locate the image of pinhole No. 2 (green) above or below pinhole Nos. 1 or 3 on the cell stream axis. Laser beam No. 2 is then positioned to intersect the cell stream centered on pinhole No. 2. Similarly, by micrometer adjustment of the 45 front surface mirror, the image of pinhole No. 3 (red) can be positioned above or below pinhole Nos. 1 and 2 along the cell stream axis. Laser beam No. 3 is then positioned to intersect the cell stream centered on pinhole No. 3. After the laser beam-pinhole alignment, the lamps are removed and sequential fluorescence measurements are performed. However, when single-laser excitation experiments are made, pinholes Nos. 2 and 3 images are adjusted to coincide with pinhole No. 1 for total or simultaneous two or three color fluorescence measurements. This method permits a single collecting lens to be used for the measurement of fluorescence from cells or particles illuminated by single or multiple laser beams spatially separated. Signals from the fluorescence sensors are amplified, integrated, and processed using hardwired analog signal processing electronics for simultaneously or sequentially occurring signals (13). An LSI-11 computer accumulates data in the LIST MODE and displays signals as pulse-amplitude frequency distribution histograms (10). To demonstrate the three-laser excitation system, exponentially growing line CHO cells and rat lung lavage cells consisting primarily of alveolar macrophages were stained with fluorochromes having separated excitation spectra and analyzed as described below. CHO cell samples were first reacted with a mouse anti-cho antibody followed by FITC labeled rabbit antimouse IgG (supplied by Dr. George Saunders), fixed in 70% ethanol, rinsed, and then stained for DNA content and total protein using Hoechst and rhodamine 640, respectively? Rat pulmonary alveolar macrophages (PAM) were exposed Ln vivo to 1.75 pm diameter green fluorescent spheres for 30 min. (15). PAM and other cells were then removed from the lungs by lavaging with saline, fixed in 70% ethanol, rinsed, and stamed for DNA content and total protein using propidium iodide (18 pg/d) and SITS (50 pg/ml), respectively. Lavage cells were also treated with RNase (40 pg/ml) at 37" C. To determine the fluorescence excitation and emission spectrum of the fluorescent dyes, calf thymus DNA was stained with Hoechst and propidium iodide and bovine serum albumin with FITC, rhodamine 640, and SITS. Stained aliquots were then analyzed on a spectrofluorometer. These results are shown in Figure 3 along with the laser line chosen for best excitation and the approximate optical filter bandwidths for the blue, green, and red fluorescence measurement channels Results With the CR-10 argon (UV), CR-4 argon (488 nm) and the CR-3000K krypton (568 nm) lasers set for sequential excitation, CHO cells were analyzed for DNA content, cell-surface antigens, and total protein (Fig. 4). The DNA content distribution (Fig. 4A) was obtained by exciting Hoechst stained cells in the UV and measuring corresponding blue fluorescence signals which served as the initial trigger for sequential fluorescence measurements (13). The first peak of the DNA content distribution represents G1 cells (2C DNA content) and the second peak G2+M cells having twice the DNA content. Cells synthesizing DNA (S phase) lie in the region between the peaks. As cells intersected the second beam (488 nm), green fluorescence from FITC-labeled antibody bound to the cell surface was recorded. The resulting distribution (Fig. 4B) is unimodal, broad, and typical of cells expressing surface antigens that are growing exponentially (18). Total protein was next measured as cells intersected the 568 nm krypton laser line and were excited to fluoresce red. The total protein distribution is also unimodal, broad and similar to that of CHO cells stained with FITC (3). Rat lung lavage cells were analyzed for DNA content, phagocytosis, and total protein by sequential excitation using the CR-3000K (530 nm), CR-4(457 nm), and CR-lO(UV) lasers, respectively, with the DNA content signal serving as the trigger or sequential fluorescence measurements. The DNA content distribution, obtained by exciting propidium iodidestained cells at 530 nm and recording red fluorescence, is shown in Figure 5A. Peak 1 represents cells having 2C DNA content and peak 2 binucleates and doublets. A very small percentage (1-4%) of cells contained between peaks 1 and 2 represent cells in S phase (5). As lavage cells intersected the second beam (457 nm), green fluorescence from phagocytized 1.75 pm diameter uniform green fluorescent particles contained in alveolar macrophages was recorded (Fig. 53). Peaks 1 to 4 represent macrophages that have phago-ytized 1, 2, 3 and 4 particles, respectively, as previously identified by cell sorting (15). Macrophages having phagocytized greater than four particles are contained between channels Computer analysis (10) of the phagocytosis data later indicated that 90% of the macrophages had phagocytized one or more Crissman HA, Steinkamp JA: Rapid, one-step staining procedures for two color analysis of cellular DNA and protein by single and dual laser flow-cytometry (submitted for publication).

4 MULTILASER FLOW CYTOMETRY 229 "BLUE - v I o LET - " LASER NO 2 EXCITATION "GREEN -YELLOW" FILTER FLOW CHAMBER FRONT VIEW) "ORANGE - RED - LOSER txc ITATIO EEAMS LBARRl ER FILTER NO 3 FIG. 2. Simplified diagram of three-color fluorescence optical sensing showing three-laser excitation scheme, collection lens, optical filter arrangement, pinholes (200 pm diameter), and photomultiplier tubes. Dichroic filter No. 2 and the front surface mirror are mounted on micrometer positioners for adjusting pinholes No. 2 and 3 spatially with respect to pinhole No. 1. Typical filter selection for lasers Nos. 1, 2, and 3 operating in the UV, 488 nm, and 568 nm, respectively: Barrier No. 1: Schott GG 400 (Schott Optical Glass Inc., Duryea, PA); dichroic No. 1: Bausch & Lomb (Bausch & Lomb Inc., Rochester, NY); shortpass No. 1: Bausch & Lomb ; barrier No. 2: Corning 3-69 (Corning Glass Works, Corning, NY); dichroic No. 2 Bausch & Lomb ; shortpass No. 2: ; and barrier No. 3: Schott RG 610. l l l l ~ l ~ I li I l I EXCITATION HOECHST PRDPlOlUM IODIDE (A) (C) FITC ANTIBODY --- GREFN SPHERES - - RHODAMINE nrn - SITS ' S, h v) w 1 0 > \ - =/ \- - WAVELENGTH (nrn) FIG. 3. Fluorescence excitation (A and C) and emission (B and D) spectra of DNA content stains Hoechst and propidium iodide bound to calf thymus DNA protein stains rhodamine 640, SITS, and FITC bound to bovine serum albumin; and 1.75 pm diameter green Fluoresbrite (Polysciences, Inc., Warrington PA) microspheres as recorded on an Aminco-Bowan Spectrofluorometer (American Inst. Co. Silver Springs, MD). The laser excitation wavelengths (arrows) are shown in A and C and the approximate fluorescence measurement regions (shaded) are shown in B and D for the three-laser, three-color fluorescence measurements of Figures 4 and 5.

5 230 STEINKAMP, STEWART AND CRISSMAN spheres. This was determined by dividing the total number of macrophages having phagocytizing one or more spheres (Fig. 5B) by the number of cells contained under region 2 of Figure 5C as described below. Total protein also was determined as rat lavage cells intersected the third beam (UV) and were excited to fluoresce blue. The protein distribution (Fig. 5C) consists of two primary regions. Peak 1 represents polymorphonuclear leukocytes, lymphocytes, and large debris and peak 2 the total number of macrophages. Discussion An advanced instrument for rapidly analyzing cells and microscopic particles using three laser beams as excitation sources of fluorescence has been described. The symmetrical placement of two of the lasers each 10" off the primary axis permitted each beam to be independently shaped and focused by separate pairs of crossed cylindrical lenses and spatially positioned within the flow cell using micromanipulators. Cell fluorescence was measured using a three-color fluorescence sensor which employed a single collecting lens. A simple external adjustment of the dichroic filter and mirror permitted either simultaneous or sequential measurement of fluorescence signals for use in single and multilaser excitation exper- UJ 1 rlaser 1 EXCITATION (UV)i 3072 f(a) I I CHANNEL NO. 1 (BLUE) DNA CONTENT LASER 2 ij r EXCITATION (488 nm) 1 m CHANNEL NO. 2 (GREEN) CELL SURFACE LASER 3 p EXCITATION (568 nm) 7 (C) CHANNEL NO. 3 (RED) YTL PROTEIN A 0 I l l l l l l l l ; l l i l l l ' CHANNEL NUMBER FIG. 4. Frequency distribution histograms of CHO cells stained for DNA using Hoechst 33342, cell-surface antigens using an FITC-labeled antibody, and total protein using rhodamine 640: A, DNA content; B, FITC-labeled cell-surface antigens; and C, protein distributions. Hoechst 33342, FITC, and rhodamine 640 bound to CHO cells were excited sequentially in the UV, at 488 nm, and 568 nm using two argon lasers and a krypton laser, respectively. LASER 1 r EXCITATION (530 nm) 1 CHANNEL NO. 3 (RED) DNA CONTENT 0 9 LASER LII W m 3 r EXCITATION (457 nm) 1 CHANNEL NO. 2 (GREEN) PHAGOCYTOSIS z o - LASER 3-1 EXCITATION (UV) I 256 r h CHANNEL NUMBER FIG. 5. Frequency distribution histograms of rat pulmonary lavage cells exposed to 1.75 pm diameter green fluorescent particles for 30 min in uiuo prior to furing in 70% ethanol and staining for DNA centent with propi&um iodide and total protein with SITS: A, DNA content; B, phagocytosis; and C, protein distributions. Propidium iodide, green fluorescent microspheres, and SITS bound to lavage cells were excited sequentially at 530 nm, 457 nm, and in the UV with a krypton and two argon lasers, respectively. iments. Various optical filter arrangements allowed color separation and blocked scattered laser light from the fluorescence channels photomultipliers. Cultured CHO and rat pulmonary lavage cells were used to demonstrate system capability. CHO cells demonstrated DNA content, cell surface antigens, and total protein measurements from three stain combinations having well separated fluorescence excitation and emission properties. Similarly, rat pulmonary lavage cells were analyzed for DNA content, phagocytosis, and protein. This data can be further analyzed using two-parameter methods for characterizing subpopulations of cells within the lung lavage sample by generating two-dimensional distributions of data stored in LIST mode (10). Although the fluorescence excitation and emission spectra partially overlapped, it was possible to make three-color fluorescence measurements by careful selection of laser excitation lines and filters with respect to excitation and emission curves. This technology has great potential in cell and particle research, and represents a new application of flow cytometry for the analysis and sorting4 of mixed cell populations using multiple markers. Sorting data not presented.

6 MULTILASER FLOW CYTOMETRY 23 1 Acknowledgments We thank G. Saunders for the FITC-labeled antibody against CHO cells; J. Wilson for assistance with phagocytosis measurements; D. Hiebert for electronics support; and G. Salzman and S. Wilkins for LSI-1 I computer software development. Literature Cited 1. Arndt-Jovin DJ, Grimwade BG, Jovin TM: A dual laser flow sorter utilizing a CW pumped dye laser. Cytometry 1: 127, Arndt-Jovin DJ, Jovin TM: Computer-controlled multiparameter analysis and sorting of cells and particles. J Histochem Cytochern 22: 622, Crissman, HA, Steinkamp JA Rapid simultaneous measurement of DNA, protein, and cell volume in single cells from large mammalian cell populations. J Cell Biol , Dean P, Pinkel D: High resolution dual laser flow cytometry. J Histochem Cytochem 26: 622, Golde DW, Byers LA, Finley T N Proliferative capacity of human alveolar macrophage. Nature 247: 373, Hulett HR, Bonner WA, Barrett J, Herzenberg LA Cell sorting: Automated separation of mammalian cells as a function of intracellular fluorescence. Science , Kamentsky LA: Cytology automation. Advances in Biological and Medical Physics. 14: 83, Melamed MR, Mullaney PF, Mendelsohn ML: Flow Cytometry and Sorting, John Wiley and Sons, New York, Mullaney PF, West WT A Dual Parameter Flow Microfluorometer for Rapid Cell Analysis. Br J Phys sec. E. 6: 1006, Salzman GC, Wilkins SF, Whitfill JA Modular computer programs for flow cytometry and sorting: the LACEL system. Cytometry 1: 325, Shapiro H, Schildkraut R, Curbelo R, Brough-Turner R, Webb R, Brown D, Block M: Cytomat R A computer-controlled muitiple laser source multiparameter flow cytophotometer system. J Histochem Cytochem 25: 836, Steinkamp JA, Fulwyler MJ, Coulter JR, Hiebert RD, Horney JL, Mullaney PF: A new multiparameter separator for microscopic particles and biological cells. Review of Scientific Instruments 44: 1301, Steinkamp JA, Heibert RD: Signal processing electronics for multiple electronic and optical measurements on cells. Cytology 2: 232, Steinkamp JA, Orlicky DA, Crissman HA Dual-laser flow cytometry of single mammalian cells. J. Histochem Cytochem 27: 273, Steinkamp JA, Wilson JS, Saunders GC, Stewart CC: Phagocytosis: flow cytometric quantitation using fluorescent microspheres. Science, in press 16. Stohr M, Eipel H, Goerttler K, Vogt-Schaden M: Extended application of flow microfluorometry by means of dual laser excitation. Histochemistry 51: 305, Van Dilla MA, Trujillo TT, Mullaney PF, Coulter JR. Cell microfluorometry: a method for rapid fluorescence measurement. Science 163: 123, Warner NL, Daley MJ, Richey J, Spellman C: Flow cytometry analysis of murine B cell lymphoma differentiation. In: Immunological Reviews, Moller G (ed.) Vol. 48, Munksgaard, Copenhagen, Denmark, 1979, p