Real-time scanning and image analysis

Journal of Immunological Methods, 109 (1988) 131-137 Elsevier

131

JIM04725

Real-time scanning and image analysis A fast method for the determination of neutrophil orientation under agarose Jens Oluf Pedersen 1, Lars Hassing 2, Niels G r u n n e t 1 and Casper Jersild 1 l Regional Centre for Blood Transfusion and Clinical Immunology, Aalborg Hospital, DK-9100 Aalborgo Denmark, and 2 Aalborg University, Institute of Electronic Systems, DK-9100 Aalborg Denmark (Received 11 September 1987, revised received 28 October 1987, accepted 30 November 1987)

We describe a newly developed method for fast determination of neutrophil chemotaxis and orientation in concentration gradients of chemotactic factors. The system implements video-based real-time scanning and image analysis of neutrophil migration under agarose, using an interactive easy-to-use computer program. Two methods for determining cell orientation are presented. No statistically significant difference between the methods was found. The analysis program distinguishes between chemokinetic and chemotactic behaviour of the cells (P < 0.01). Key words: Neutrophil; Chemotaxis; Chemokinesis; Polarization; Cell orientation; Image analysis

Introduction

Chemotaxis essentially means the ability of cells to orient in concentration gradients of certain factors in their environment. Assay systems for the detection of neutrophil orientation and polarization have previously been described by Zigmond (1977, 1978 and 1981). These techniques permit the study of individual cells and make time-lapse photographs possible which in turn is the basis of determining cell orientation and speed. Biiltman and Gruler (1983) and Gruler and Btiltman (1984) presented a thorough analysis of neutrophil movement based on series of photographs of small populations of cells in a Zigmond orientation

Correspondence to: J.O. Pedersen, Department of Medicine and Nephrology C, Aalborg Hospital, DK-9100 Aalborg, Denmark.

chamber. They found that the average degree of orientation of one single moving granulocyte over a period of time equalled that of a population of cells at any one time. The under-agarose system for granulocyte chemotaxis has been shown to give reproducible results in studies evaluating migration distance. Granulocytes migrating under agarose exhibit a characteristic shape and show polarization of the nuclear material when challenged with a concentration gradient of chemotactic factors (Palmblad et al., 1982; and unpublished observations). The aim of this study was to develop an improved system for measuring chemotactic parameters including cell orientation. We describe in this paper a video-based image analysis system, which includes computer hardware and software designed to extract these parameters from granulocyte migration under agarose using two different algorithms. An analysis of granulocyte orientation in 15 healthy blood donors is presented.

0022-1759/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

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Materials and methods

Cell separation Granulocytes were purified from EDTA-stabilized whole blood using Hypaque/Ficoll discontinuous gradient centrifugation as described by Boyum (1976) with the modification of Ferrante and Thong (1978). Cells were harvested and washed three times in RPMI 1640 (Sigma cat. R-6504). Cell counts were adjusted to 2.5 x 107 granulocytes/ml. Migration assays Migration experiments were performed using an assay system for granulocyte migration under agarose (Nelson et al., 1975). The gels were made from 0.75% w / v agarose (Litex HSA, no. 4431, from Litex Industries, Glostrup, Denmark) moulded on gelatmized microscope object slides. The agarose was suspended in medium containing minimal essential medium (Gibco cat. no. 3301430) as tissue culture medium. The buffer system was Hepes (Gibco cat. no. 043-5630D) at a final concentration of 0.05 mol/1 (pH adjusted to 7.1 using 1 N NaOH). Gelatin (Sigma cat. 0510) was added to the agarose gel at a final concentration of 0.25% w / v as a source of protein (Chenoweth et al., 1978). Chemotactic systems Chemotactic systems comprised three wells (diameter 2.5 mm) punched out in the agar gel opposite each other at a mutual distance of 2.5 nun. 10/~1 of cell suspension (2.5 X 105 cells) were transferred to each well in the middle row. The chemoattractant used was N-formyl-methionylleucyl-phenylalanine (N-FMLP, Sigma cat. F3506) at a concentration of 10 -8 mol/1 medium RPMI 1640. 10/~1 of chemoattractant were transferred to one well, 10 ffl of culture medium RPMI 1640 were used as control in the other well. For the study of chemokinesis, N - F M L P was mixed with the liquid agarose medium at 56 ° C to a final concentration of 10 -9 mol/1 before moulding the assay plates. 2.5 x 105 granulocytes were transferred to the cell well. The agarose plates were incubated at 37 ° C for 120 min in humid, atmospheric air. To ensure preservation of cell shape and nuclear polariza-

tion, the migration was stopped by floating the agarose plates in 2.5% glutaraldehyde in phosphate-buffered saline. After fixation for 1 h, the agarose gels were removed, the slides stained with Wright's stain and provided with a coverslip mounted in Eukitt.

Microscope and video scanner A Leitz Dialux microscope provided with a charge coupled device (CCD)-based video camera (Sony DXC-101p) in the photo tube was used. The objective was x 25 for all studies. Computer The basic features of the computer hardware used are as follows. It has been built around a VME bus linking two boards together: a 12.5 MHz central processor unit (CPU) board from Eltec, F.R.G., which uses the '68000' microprocessor from Motorola and a video scanner/ graphic board designed to the system. The computer holds two 51 in disk drives, each providing 0.9 megabytes of background storage. The operating system is O S / 9 and the memory capacity of 1 megabyte RAM, is partly formatted as RAM diskette for fast handling of program and images. Images captured from the camera, stored images as well as the grey level histograms, angle distribution histograms and other results are displayed on a black and white video monitor with a resolution of 256 x 256 pixels. A graphics dot matrix printer (Facit 4513) permits printing of screen dumps of images, histograms and results. The analysis program is written in the language 'C' to provide fast execution. Although the computer was made for the system described, the software will run on other systems as well. The computer described herein shares features with a number of commercially available machines, especially those made for image processing purposes. Image analysis in the sense described here requires a certain minimum of computer power if it should be an effective scientific and clinical tool. However, in the appendix we give the name and address of a company which produces image analysis equipment (software and hardware) for many purposes. The easiest way to access our programs is to contact this company directly.

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(6) Perform calculations of the orientation index P(1). (7) Print out the results, digitized pictures of cells and angle distribution histogram (Figs. 3 and 4). Notes: (a) Method 1 defines the gravity midpoint of the nucleus of each cell and computes the point on the cytoplasmic border where the distance to the gravity midpoint is maximal. The straight line defined by these two points is the orientation axis of the cell (Fig. 2A). The direction of movement of each cell is determined by the vector from the nucleus towards the farthest point on the cell border. The angle between this axis and the axis of the chemotactic gradient is calculated. The cosine to this angle is determined and the mean for the cell population in the field is calculated. This mean value is the orientation index P(1). Method 2: Every pixel point within the area of each cell has co-ordinates in relation to the screen. The best fit of a straight line through these points is computed for each cell according to the method of least squares. This line is

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Fig. 1. Grey level histogram resulting from analysis of a population of 52 granulocytes migrating under agarose towards a gradient of N - F M L P . x-axis: The grey level range. Vertical dotted lines indicate boundaries for thresholding the image into three grey levels, y-axis: n u m b e r of pixels of each grey level (logarithmic scale).

They will be able to deliver our programs on disk with the necessary modifications for different types of hardware. Experienced computer programmers will, based on the essential ideas given here in the thresholding procedure and the orientation algorithms, be able to construct similar programs to existing systems. Steps in image analysis program The menu-driven program runs interactively with the operator, using a 'mouse'. The following steps are used to complete the analysis of a stained slide: (1) Load program into computer. (2) Select method to be applied in determination of orientation (method 1 or 2, cf. note (a) below). (3) Select by light microscope the area to be analyzed. (4) Adjust light intensity and contrast level, using the grey level histogram displayed in real-time on the image monitor to achieve the widest possible grey level range (Fig. 1). (5) Initiate program to define cell and nuclear boundaries (cf. note (b) below). The final part of this program shows the orientation axis of each cell on the screen (Fig. 3).

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Fig. 2. Determination of the orientation axis of a granulocyte. A: Illustration of orientation axis and angle between this and the chemotactic gradient according to method 1. B: Illustration of granulocyte orientation axis and angle according to method 2 (see text for description). '0 o, is the chemotactic gradient axis, ' a ' indicate the angle between the gradient axis and the cell axis.

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Fig. 3. Printout from dot matrix printer (screen-dump) of digitized, thresholded image of granulocytes migrating under agarose towards a concentration gradient of N-FMLP. White lines within the cells indicate the computed orientation axis of each individual granulocyte.

the orientation axis of the cell. The position of the pixels defining the nucleus in relation to the cytoplasmic area is determined. The direction of movement is defined, assuming that the nucleus is positioned at the posterior of the cell. The cosine to the angle between the gradient axis and the orientation axis is computed for each cell in the field, and the mean is calculated. This mean value is the orientation index P(1). (b) Cells located too close to be identified as separate cells may be divided on the screen by the operator using the 'mouse' or can be ignored if too complex. When cell separation is considered correct it is confirmed by the operator. After a few seconds the digitized, thresholded image of cells with the orientation axis of each cell is shown on the screen (Fig. 3). The orientation axes are now either confirmed or may be corrected by the operator. After confirmation, the angle distribution histogram appears instantly (Fig. 4) together with the orientation index P(1) and the mean angle of orientation in degrees.

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Fig. 4. Angle distribution histogram for a population of granulocytes (n = 52) migrating under agarose towards a gradient of N-FMLP. Zero degree is the direction towards the concentration gradient; +180 degrees indicates the opposite direction.

Investigations Chemotactic and chemokinetic studies were performed using granulocytes from 15 healthy blood donors. These scannings were all done at the leading front of cells in the gradient axis. Only fields containing at least 30 cells were taken into consideration. To compare the two different methods for estimating cell orientation 15 different granulocyte images were scanned and analyzed using the two methods consecutively on the same image. Variation in the scanning procedure was estimated scanning the same microscope image ten times, using the two different algorithms.

Stat&tics Wilcoxon's test for paired differences was used to compare methods 1 and 2. To compare chemokinetic and chemotactic migration the MannWhitney test was performed. A P value below 0.05 was considered significant.

Results

Image analysis A characteristic grey level histogram is shown in Fig. 1. The histogram was obtained from grey

135 TABLE I

TABLE II

COMPARISON OF ORIENTATION INDICES, P(1), DERIVED BY TWO DIFFERENT ALGORITHMS (METHOD 1 AND 2) APPLIED TO IMAGE ANALYSIS OF UNDER AGAROSE MIGRATION ASSAYS OF NORMAL CONTROL GRANULOCYTES

GRANULOCYTE CHEMOTAXIS AND CHEMOKINESIS UNDER AGAROSE IN 15 HEALTHY INDIVIDUALS

There was no statistically significant difference (P > 0.1) between the two methods. Image no.

Method 1 P(1)

Method 2 P(1)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

0.463 0.392 0.365 0.365 0.001 0.255 0.547 0.511 0.244 0.039 -0.117 - 0.342 0.492 0.305 0.320

0.457 0.365 0.386 0.458 0.057 0.268 0.542 0.607 0.323 0.026 -0.113 - 0.170 0.388 0.308 0.358

level analysis of the original cell population shown as a printout in Fig. 3. The histogram has a tri-phasic distribution of grey levels f r o m light to dark corresponding to background, cytoplasma and the nuclei. Fig. 3 shows a printout of the digitized and thresholded (i.e., the total grey level range is transformed into black, grey and white) image of granulocytes oriented in a gradient of N - F M L P . The main part of the cells are polarized since the nuclear material is located at one end of the granulocytes. The orientation axis of each cell is shown according to m e t h o d 1. The angle distribution histogram for the cell population scanned is shown in Fig. 4. The cells orient towards zero degrees (which is the gradient axis) with a Gaussian-like distribution of angles away from the zero degree axis.

Comparison of methods 1 and 2 for cell orientation Table I shows P(1) values of 15 different images obtained by methods 1 and 2. There was no statistically significant difference between the two methods ( P > 0.1), although there was a tendency

Orientation index P(1) according to method 2: Data are provided as medians with interquartile ranges. The difference between chemotactic and chemokinetic migration was statistically significant (P < 0.01). Chemotaxis (n =15): Chemokinesis ( n = 15):

0.423 (0.357-0.541) 0.085 ( - 0.134-0.089)

for m e t h o d 2 to exhibit higher orientation indices than did m e t h o d 1.

Chemotaxis and chemokinesis Table II shows the orientation indices of granulocytes from 15 healthy blood donors in chemotactic and chemokinetic assays. In chemotactic assays, the orientation index P(1) was 0.423 (0.357-0.541) (median and interquartile range). Chemokinetic assays gave a median P(1) of 0.085 with an interquartile range from - 0 . 1 3 4 to 0.089. The difference between the chemotactic and chemokinetic assays was statistically significant ( P < 0.01) using the M a n n - W h i t n e y test.

Variation Scanning one image ten times consecutively revealed a coefficient of variation CV --- 0.101 for algorithm 1 and CV = 0.049 for algorithm 2 for a m e a n P(1) = 0.351 and 0.372 respectively.

Discussion The idea of using electronic equipment for the analysis of leukocyte migration is not new. Different forms of optokinetic and cinematographic methods have previously been described b y others (Valerius et al., 1977; Dahlgren et al., 1979; Moss et al., 1979; C h e u n g et al., 1982; Mahler et al., 1984). The study of leukocyte migration is complicated b y the question of whether the cells have m o v e d in a directional m a n n e r (chemotaxis) from the starting point or rather in a r a n d o m manner, but at a higher speed (chemokinesis).

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Many assay systems widely used for the determination of granulocyte migration do not measure the essential parameter of chemotaxis namely the degree of orientation in the chemotactic gradient. Time-lapse studies of single cell migration and orientation have been previously described using a modified under-agarose assay system (Coates et al., 1985; Burton et al., 1986; Donovan et al., 1987). Recently, a system using real-time analysis to follow the tracks of rabbit neutrophils and other cells while migrating in filming chambers has been described (Dow et al., 1987). During migration under agarose, human neutrophils show polarization with localization of their nuclear material to the rear of the cell. Such changes may provide information about the direction of movement and it is possible to preserve cellular shape and nuclear polarity by fixing the preparations with glutaraldehyde. The algorithms for the determination of orientation of cell populations measured at one time as described in this study are based on such observations. Using the fast running computer program we describe here, a full image analysis for the determination of the orientation index P(1) and an angle distribution histogram takes only a few minutes. The two algorithms described for the estimation of the orientation index P(1) gave results comparable to those obtained by others (Donovan et al., 1987) using time-lapse studies. Method 2 showed less variation (CV = 0.049) compared to method 1 (CV = 0.101). This may have been due to the fact that all pixel points within the area of each cell were taken into consideration in the determination of the orientation axis thus taking advantage of all the information available. This method also clearly distinguished between chemokinetic and chemotactic behaviour of the cells as shown in Table II. A major problem applying image analysis to populations of granulocytes is their tendency to stick together upon stimulation with chemotactic factors. Also, when too many cells migrate in the chemotactic field, it may be difficult to separate individual cells. These problems can partly be solved by decreasing the number of cells in the cell well. 10 6 granulocytes in the well normally gives too crowded an image to analyze success-

fully. We found, that 2.5 × 105 granulocytes gave satisfactory chemotactic and chemokinetic responses as well as permitting the analysis of single cells. Furthermore, cells sticking together can be divided by software routines when this is appropriate. Impurities in the scanning field (i.e., objects too small to be recognized as a cell) are discarded automatically by the program and removed from the screen during the thresholding procedure. The under-agarose assay we describe here does not produce a linear front of chemotactic gradients since chemotactic factors diffuse radially from a circular well. Parallax displacement might introduce errors into the calculations of P(1). Using our analysis system, the operator is warned about such errors if the angle distribution histogram in chemotactic assays is asymmetric with the top point beside the zero degree system axis. Another advantage of the system described is that it permits up to six chemotactic or chemokinetic assays to be performed simultaneously on the same slide, thereby minimizing variation between assays. In summary, based on observations of granulocyte morphology and nuclear polarization during migration under-agarose, we have applied fast running image analysis to fixed preparations. The two algorithms for estimating granulocyte orientation presented here gave reproducible results, and were equally useful. It is possible to extract the essential parameter of chemotaxis, namely the degree of orientation of cells, from the under-agarose assay without any modification other than reducing the cell number to 2.5 × 105/well. Image analysis of granulocyte chemotaxis is proving to be very useful in the investigation of patients with inflammatory and infectious diseases.

Appendix Image analysis systems, which share technical features with our system are commercially available from: Scan Beam Ltd., Rosendalsvej 17, DK9560 Hadsund, Denmark (Telephone: 458571599). This company is also in a position to provide the programs described here. Questions concerning software compatibility with existing systems, and

137 p o s s i b l e m o d i f i c a t i o n s s h o u l d a l s o b e a d d r e s s e d to the company.

Acknowledgements T h i s w o r k was s u p p o r t e d b y g r a n t s f r o m t h e Aalborg voluntary blood donors foundation, the Aalborg Municipal fund for medical research, and the Aalborg Discount Bank study and research foundation. T h e skilful t e c h n i c a l a s s i s t a n c e o f M r s . ElseM a r i e O v e r b y w a s i n v a l u a b l e . J. G u s t a f s o n , H. N i e l s e n a n d K . W . F a b r i n , A a l b o r g U n i v e r s i t y , are g r e a t f u l l y a c k n o w l e d g e d for t h e i r c o m p e t e n t e n g i n e e r i n g a n d a d v i c e in c o m p u t e r c o n s t r u c t i o n and programming.

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Donovan, R.M., Goldstein, E., Kim, Y., Lippert, W., Kailath, E., Aoki, T.T., Cheung, A.T., Miller, M.E. and Chang, D.P. (1987) A computer-assisted image-analysis system for analyzing polymorphonuclear leukocyte chemotaxis in patients with diabetes mellitus. J. Infect. Dis. 155, 737. Dow, J.A.T., Lackie, J.M. and Crocket, K.V. (1987) A simple microcomputer-based system for real-time analysis cell behaviour. J. Cell Sci. 87, 171. Ferrante, A. and Thong, Y.H. (1978) A rapid one-step procedure for purification of mononuclear and polymorphonuclear leukocytes from human blood using a modification of the Hypaque/Ficoll technique. J. Immunol. Methods 24, 389. Gruler, H. and Biiltman, B.D. (1984) Analysis of cell movement. Blood Cells 10, 61. Mahler, J., Martell, J.V., Brantley, B.A., Cox, E.B., Niedel, J.E. and Rosse, W.F. (1984) The response of human neutrophils to a chemotactic tripeptide (N-formyl-methionyl-leucylphenylalanine) studied by microcinematography. Blood 64, 221. Moss, V.A., Simpson, H.K.L. and Roberts, J.A. (1979) A semi-automatic method of measuring leukocyte movement. J. Immunol. Methods 27, 293. Nelson, R.D., Quie, P.G. and Simmons, R.L. (1975) Chemotaxis under agarose: A new and simple method for measuring chemotaxis and spontaneous migration of human polymorphonuclear leukocytes and monocytes. J. Immunol. 115, 1650. Palmblad, J., Uden, A.-M., Venezelos, N. and Afzelius, B. (1982) Neutrophil migration and orientation under agarose: Findings in patients with the immotile cilia syndrome and effects of cytochalasin B and vinblastine. Adv. Exp. Biol. 141, 49. Valerius, N.H. (1977) Neutrophil granulocyte chemotaxis in vitro. Comparison of the response to casein and a bacterial chemotactic factor, and evaluation of an automatic method for counting cells on a membrane filter surface. Acta Pathol. Microbiol. Scand. Sect. C 85, 289. Zigmond, S.H. (1977) Ability of polymorphonuclear leukocytes to orient in gradients of chemotactic factors. J. Cell Biol. 75, 606. Zigmond, S.H. (1978) A new visual assay of leukocyte chemotaxis. In: J.I. Gallin and P.G. Quie (Eds.), Leukocyte Chemotaxis. Raven Press, New York, p. 57, Zigmond, S.H., Levitsky, H.I. and Kreel, B.J. (1981) Cell polarity: an examination of its behavioral expression and its consequences for polymorphonuclear leukocyte chemotaxis. J. Cell Biol. 89, 589.