A fluorescence microscope attachment for flow-through cytofluorometry

Printed in Sweden Copyright 0 1974 by Academic Press, Inc. AN rights of reproduction in my form reserved

Experimental Cell Research86 (1974) 53-58

A FLUORESCENCE

MICROSCOPE

ATTACHMENT

FOR FLOW-THROUGH

CYTOFLUOROMETRY G. von SENGBUSCH and B. HUGEMANN Battelle-Institut e. V., Frankfurt am Main, BRD

SUMMARY A flow-through systemfor rapid automated analysis of intracellular fluorescenceparametersis described.In combination with commercially available instrumentation for fluorescencemicroscopy, fluorometry and pulse height analysis, the hydrodynamic focusing flow-through principle can be successfullyapplied. The specificadvantagesand disadvantagesof this principle compared with the mechanicalfocusing systemare discussed.

Some equipment for rapid automated cytofluorometry is already in use at different laboratories [l-5] and has been commercially available for several years (Cytofluorograf, Bio/Physics Systems Inc., Mahopac, N.Y., USA; Impulscytophotometer ICP, Phywe, Giittingen, BRD). However, at least in addition to the compact instruments which have been designed for special purposes, other, more versatile attachments should be made available in order to extend the use of conventional microscope fluorometer equipment to include rapid automated microfluorometry. In the following, an instrumentation for rapid automated microfluorometry is discussed, based on the flow-through principle and on a conventional microscope fluorometer equipped with a simple flow-through chamber and the necessary electronic equipment. Two typical examples illustrating the application of this instrumentation are described.

Considerationsfor seleetiagthe Iaminar-flow principle as a basis for the flow-through teeladqae proposed

Two different techniques have proved useful for the automated identification of cells or particles based on the flow-through principle: - the mechanical focusing system [6], where the cell suspensionis added through a vertical capillary (arranged in the optical axis of the microscope) to a stream of cell-free liquid flowing perpendicular to the microscope axis (fig. 1). The microscope is focused on the outlet of the capillary tube. All cells must pass the focal plane and are then washed away by the transverse flow. In the focal plane the cell fluorescence is excited by means of light incident through the microscope objective. The fluorescence light is collected by the same objective, then passesthe dichromatic mirror and finally reaches the photomultiplier; - the hydrodynamic focusing system where the cell suspension is centrally introduced to Exptl Cell Res 86 (1974)

54 von Sengbusch and Hugemann

time-dependent intracellular turnover rates) does not decrease the signal-to-noise ratio markedly. + emission filter I The use of the mechanical focusing system is often limited due to either primary cell dichromatic mirror agglutination (becauseof incompletely separated cells) or to secondary cell agglutination (caused by a reaction of the cells to be measured). Therefore, rather large diameters of the capillary tube (about 100 to 150 pm) are necessaryto avoid clogging. In the case of the hydrodynamic focusing sample system the outlet of the central injection :nlet capillary can be of appropriate size (>250 Fig. 1. Schematic diagram of the optical part of an ,um) since the central flow is hydrodynamiautomated flow-through cytofluorometer with injection of the cell suspension parallel to the optical axis cally narrowed down to 30 to 50 pm). Cell of the microscope and perpendicular to the cell-free clots may cause the central flow to leave the laminar stream, applying the principle of “mechanical focusing”. stable position for a very short time but the central flow stabilizes again within millia laminar stream flowing at right angles to seconds. The light signal reaching the photomultiplithe optical axis of the microscope [2]. If appropriate flowing conditions are chosen, er cathode includes the cell fluorescence and the central flow can be focused hydrodynami- the background signal of the total measuring cally. The fluorescence emitted by the cells area. In the case of the mechanical focusing can be taken up at different directions, i.e. system the diameter of the capillary tube and incident, in the direction of transmission, or consequently also the diameter of the measurat an angle of 90°C. Fig. 2 shows the measur- ing area limited by the measuring diaphragm ing arrangement based on the hydrodynamic focusing system. As the central flow remains stable, severalmeasuring areascan be arranged photomultiplier in series. This possibility has led to the development of special arrangements for cell se+ emission filter paration [7]. Considering the particular measuring problem to be solved, the hydrodynamic focusing system proved to be more advantageous mainly for the following reasons: objective - Cell agglutination due to different reactions (for instance, measurement of the binding of FITC-labeled conA to cells) and 2. Schematic diagram of the OPtical part of an big cells up to a diameter of 50 pm do not Fir. automated flow-&rot& cytofluorometer Gith injeccause obstruction of the flow-through system. tion of the ceil suspension perpendicular to the optical of the microscope and parallel to the cell-free - Background fluorescence of the suspension axis sheath flow, applying the principle of “hydrodynamic medium (for instance during measurement of focusing”. photomultiplier

zry&if~

&&i-f dichromtic mirror

Exptl Cell Res 86 (1974)

Automated cytofluorometry

55

.-sin. d.

Fig. 3. Design drawing of the flow-through chamber schematically illustrated in fig. 2. s.in., sample inlet; skin., sheath flow inlet; out., outlet; r.ud., radial adjustment; aad., axial adjustment of the injection capillary (bzJcap.); skcap., sheath capillary.

is about ten times larger than the cell diameter. Therefore, the background signal remaining almost constant may exceed many times the signal of the cell fluorescence. However, expressed in terms of electronics, the background signal is a “d.c.” or a very slowly varying signal, whereasthe fluorescence signal of the cell is an “a.c.” signal of high frequency. This means that these two signals can be separated electronically by means of an a.c. amplifier. Nevertheless, a high “dc.” signal also causes an “a.c.” noise in the photomultiplier and consequently reduces the signal-to-noise ratio of the “a.c.” signal. For these reasons the measuring area should be as small as possible. This condition is satisfied with the hydrodynamic focusing system since the diameter of the measuring area must not exceed the central flow diameter perpendicular to the flow direction and about two cell diameters parallel to the flow direction. Compared with the mechanical focusing system the signalto-noise ratio can be improved significantly by use of the hydrodynamic focusing system (up to a factor of 10) if the suspension medium shows high background fluorescence.

Flow-through system

The flow-through chamber has the following parts (fig. 3): - The central capillary tube with an outside diameter of 0.6 mm and an inside diameter of 0.34 mm (injection capillary). - The sheath capillary tube made of quartz glass. - The holding devices for the central and the sheath capillary tube. - The adjusting device for the central capillary tube. The central capillary tube can be precisely adjusted both in radial and in axial direction. The overpressure at the inlet of the sheath capillary tube is maintained at a level of 20 mmHg. The overpressure at the inlet of the central flow capillary tube is kept between 10 and 20 mmHg and is controlled by means of a sample container that can be fixed at different levels. Optical elements of the flow-through microfluorometer

The flow-through chamber can easily be fixed on the X, Y stage of a fluorescence Exptl CeZl Res 86 (1974)

56 van Sengbusch and Hugemann

microscope with incident light path (Leitz) as schematically shown in fig. 2. The light path for the fluorescence light between dichromatic mirror and photomultiplier can be simplified significantly compared with conventional microscope photometer attachments, becauseit is not necessary to watch the measuring field limited by the measuring diaphragm and the measuring aperture can be kept constant. Including the lens (50 mm focal length) for focusing the rear focal plane of the objective onto the photocathode of the photomultiplier all optical elements between the light source, the object plane and the photomultiplier are UV-transmittant (e.g. for measuring the primary fluorescence of proteins).

Electronic analysis of the fluorometric signals

According to the size of the measuring field and the velocity of the central stream, the cells pass the measuring field within 50 to 300 ,usec. The current pulses generated in the photomultiplier are converted to voltage pulses by means of a current voltage converter which has a load resistance of 20 kHz. The upper limiting frequency of the electronic system can be varied between 2 kHz and 70 kHz so that an optimum signal-to-noise ratio can be selected for a given pulse duration. The amplification can also be varied in calibrated steps between 10 and 320 times in order to compensate for pronounced changes of fluorescence intensities due to a timedependent reaction (e.g. fluorogenic substrate turnover). The pulses are analyzed by means of a pulse height analyzer (LABEN) equipped with a modified analogue/digital converter for pulses of 50 ~1sto 1 msec duration with an analyzing time that can be preselectedbetween 100,usecand 2 msec.The stored signals are fed into an X-Y recorder Exptl Cell Res 86 (1974)

where they show directly the histogram of the pulse height distribution (i.e. the histogram of the fluorescence per particle or cell). APPLICATIONS

DNA-histogram of nuclei from mow liver

The DNA histogram of mouse liver nuclei as measured by the flow-through microfluorometer described above is shown in fig. 4. The suspension of nuclei was fixed in methanol (abs.) (15 min) and then stained in ethidium bromide solution (1O-3 mol/l in Hanks solution) for 20 min. After having been stained the nuclei suspension was washed again in Hanks solution and did not show any essential alterations when stored for not more than one week at 4°C. Manual measurementson 200 nuclei taken from such a suspension showed the following results: - coefficient of variation of the diploid peak 8.5 % - coefficient of variation of the tetraploid peak 7.0 %. The corresponding coefficients of variation of the histogram measured with the hydrodynamically focusing system are 8.5 % and 6.5 %, respectively (fig. 4). Histogram of intracdlular FDA turnover

Fig. 5 shows the frequency distribution of the intracellular fluorescence intensities per cell in L cells, 2, 7.5 and 25 min after fluorescein diacetate (1O-s mol/l) was added to the suspension medium (MEM, pH 7.0), measured by use of the system described above. FDA being non-fluorescent itself penetrates into the L cells and is intracelhtlarly hydrolysed by esterases, thus forming the highly fluorescent product fluorescein [8, 91. The time-dependent reactions of the intracellular

Automated cytofluorometry

57

2.10

1.10'

0

5 a

,-: GyL channel

enzymatic turnover of non-fluorescent substrates into fluorescent products are expected to provide information on intracellular enzyme activity and permeation properties of the cell’s outer membrane [9-l 11. DISCUSSION The coefficient of variation of 6.5 % as shown in fig. 4 is in accordance with the coefficient of variation of 7.0 % found in manual control measurements of the suspension of nuclei used. As no preparation with a known coefficient of variation of less than 6.5 Y! has been available the actual resolution of the arrangement was not directly tested.

lea

Figs 4-5. Abscissa: rel. fluorescence intensity/cell; ordinate: cells/channel. Fig. 4. a, Frequency distribution of the DNA content/nucleus measured on a nuclei suspension from mouse liver. The histogram was obtained from 3 x 1W nuclei using a flow rate of IO’ nuclei/min; b, enlarged by the factor 5. Fig. 5. Frequency distribution of the intracellular fluorescence intensity per cell in L cells a, 2 min; b, 7.5 min; c, 25 min after fluoresceindiacetate (FDA) was added to the suspension medium (MEM, pH 7.0). The curves are normalised for equal cell number. The histograms were measured at a flow rate of lop cells/min.

When objectives with high numerical aperture (up to 1.25) are used, the optical conditions for excitation of fluorescence are not constant throughout the total cross section of the central flow becausethe exciting beam forms a cone with a large aperture angle. According to the measurement of fluorescence intensity of a fixed cell situated in the axis of the central steam during systematic defocusing, the light intensity varies about 10% throughout a central stream of for instance 30 ,um diameter using an objective with a numerical aperture of 1.00. For these reasons, the hydrodynamic focusing system has in the past generally been used in combination with a laser [l, 51. Exptl Cell Res 86 (1974)

58 von Sengbusch and Hugemann

Because of the great light flux per unit area in the laser beam a fluorescence signal of sufficient size can be obtained even if the aperture used for excitation is almost zero. In this case the optical conditions for excitation are almost constant throughout the cross section of the central stream, and distributions with coefficients of variation in the range of 3 % can be resolved. Therefore, a continuous dye laser with frequency doubling and adjustable wavelength would be an ideal light source for this purpose. As such a light source is still rather expensive and not very stable, usually argon lasers are used for excitation of fluorescence. However, the wavelength of this type of laser is only variable in discrete steps above 3,= 350 nm, it has a gap between 360 and 450 nm, and can be used best in the range between 450 and 550 nm. In the case of the mechanical focusing system the problem of achieving homogeneous excitation conditions throughout the central stream of cell suspension does not exist. Even with objectives of high numerical aperture practically homogeneous excitation conditions can be achieved in the focusing plane through which the cells must pass parallel to the optical axis before they are swept away by the cell-free laminar stream. Distributions with a coefficient of variation of less than 5 % can be measured, The mechanical focusing system therefore has the advantage that conventional light sourcescan be used without reducing the resolution of histogram peaks. However, in many cases this advantage cannot be fully utilized becauseof the serious difficulties pointed out on p. 53-54. Thus, taking into account a slight decrease in the resolving power, the hydrodynamic

Exptl Cell Res 86 (1974)

focusing system can be successfully applied by using conventional light sources and fluorescence microscopes with high numerical aperture of the exciting beam and for the uptake of the fluorescence emitted. In our laboratories this arrangement is also usedin combination with a two-colour analysing system consisting of a microscope binocular tube, a dichromatic mirror as beam splitter and two photomultipliers. Using this system, the fluorescence can be measured at two different wavelengths. For cell volume measurements a modified Coulter Counter system as described by Crisman & Steinkamp [12] could be attached to the microfluorometer described. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Sernetz,M, Fluorescence techniques in cell biology (ed A Thaer & M Semetz) p. 255. Springer, Heidelberg (1973). Mtiller, 6, 1von’ Sengbusch, G & Sernetz, M, Exptl cell res. Submitted for publication. Van Dilla, M A, Trujillo, T T, Mullaney, P F & Coulter. J R. Science 163 (1969) 1213. Karnenisky, L A, Cytolo& autbmation (ed D M D Evans) p. 177. Livingstone, Edinburgh, London (1970). G&de, W, Fluorescence techniques in cell biology (ed A Thaer & M Semetz) p. 79. Springer, Heidelberg (1973). Biihm, N, Sprenger, E & Sandritter, W, Fluorescence techniques in cell biology (ed A Thaer & M Sernetz) p. 67. Springer, Heidelberg (1973). Holm, D M & Cram, L S, Exptl cell res 80 (1973) 105. Bonner, W A, Nulett, H R, Sweet, R G & Herzenberg, L A, Rev sci instr 43 (1972) 404. Rotman, B& Papermaster, B W, hoc hat1 acad sci US 55 (1966) 134. Von Sengbusch; G. In preparation. Rotman, B, Fluorescence techniques in cell biology (ed A Thaer & M Sernetz) p. 333. Springer, Heidelberg (1973). Crissman, H A & Steinkamp, J A, J cell biol 59 (1973) 766.

Received January 22, 1974