ELSEVIER

Journal

of Microbiological

Methods

26 (1996)

11-20

Journal ofMicrobiological Methods

Measurement of filamentous cyanobacteria by image analysis

Anthony
Received

E. Walsby*, Avril Avery

8 June 1995; accepted 5 October 1995

School of Biological Sciences, University of Bristol, Woodland Road, Bristol, BSS IUG, UK

Abstract
A method of image analysis is described for measuring total filament lengths of cyanobacteria collected on membrane filters from samples of natural populations or cultures. Images of autofluorescent filaments viewed by epifluorescence microscopy were transferred to a computer screen by using a high resolution television camera and an image integrator to enhance sensitivity. The digitised images, saved as 0.4 Mbyte bitmap computer files, were analysed with an image analysis program. An evaluation is made of procedures for determining the total length of filaments from the detected area or perimeter of their skeletonised images. Methods are described for correcting errors arising from the orientation, crossing and overlapping of filaments.
Keywords:

Image analysis; Cyanobacteria;

Filaments

1. Introduction Light microscopy provides a means of distinguishing and enumerating different microorganisms in natural populations. The biomass of unicellular organisms can be estimated by counting and measurements of cell volume but there are problems in applying these methods to filamentous organisms, which exhibit large variations in length. It is difficult to measure the lengths of filaments directly with microscope graticules because the filaments are often curved and oriented at all angles. Olson [l] described a method of estimating filament length that is based on the number (11) of intersections that filaments make with an overlying grid: the filament length is given by nid4, where i is the mesh interval on the grid. The method assumes that filaments are random-

*Corresponding

author.

ly oriented, so that all angles are equally represented in large samples. Olsons method has been used quite widely for measuring the filament length of algae and cyanobacteria in cultures [2-41 or natural populations [5,6] and its application to such systems has been checked empirically by Bott and Brock [7]. Another method based on a grid was described by Bailey-Watts and Kirika [S] who emphasized that the size of the grid squares should be small in relation to the length of the image of the filament. This requirement is fulfilled by using video images, which comprise a fine grid of pixels that can be analysed by computer. Sheath et al. [9] described a system that involved making measurements on video-images from photographs of individual filaments of Batrachospermum sp. with the cursor of a digitising tablet (see also [lo]). Hoogveld and Moed [l l] used a computer to determine the lengths of individual filaments of Oscillatoria sp. traced with the image of a cursor on a digitising tablet projected with a

0167-7012/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved PII SO167-7012(96)00816-O

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A.E. Walsby, A. Avery I Journal

of Microbiological

Methods 26 (1996) 11-20

drawing tube into the image plane of the microscope. Each of these methods requires manual interaction of the cursor with the images of individual filaments; this impairs the precision and limits the speed with which images can be analysed. We describe an automated method of determining filament length by computer image analysis. It entails transferring microscope images of filament arrays from a video camera to a computer with a framegrabber system. The system incorporates an integrator that increases image brightness and contrast; this obviates the need to use intermediate photographic images even when analysing faint objects illuminated by epifluorescence. The positions of filaments are located with a computer software system that distinguishes lines of pixels within selected grey levels. Images of spurious objects can be eliminated by editing functions. The lengths of filaments can then be determined from measurements made on the selected arrays of pixels, but to do this it is necessary to understand the principles and potential errors of the image analysis system. This method is much faster than previous methods and more accurate than Olsons grid intersection method. We have used it for determining the total length of Anabaena filaments from cultures and Oscillutoriu filaments in samples of known volumes of lake water. We used an epifluorescence microscope system in which cyanobacteria fluoresced strongly, so that these organisms could be distinguished in the presence of other algae, which autofluoresce only weakly. The method can be applied to other phytoplankton by rendering them fluorescent with primuline yellow [12] or used with bright field illumination.

of Anubuena (PCC 9332). 2.2. Filtration

Jlos-uquue

CCAP

strain

1403f 13f

Samples of cultures and of lake water were filtered with cellulose acetate membrane filters (which remain flat on drying) of pore size 8 pm and 50 mm diameter (Filter AE 99, Schleicher and Schuell). The membrane filters were air dried overnight and then wrapped in aluminium foil envelopes. Filaments were fragmented if subjected to undue contact pressure but they were undamaged by contact with the foil and during storage over several years. Because some of the light fluoresced by filaments is reflected from white membrane filters, producing halos around the filament images, black-stained filters are recommended for epifluorescence microscopy [ 13,141. The halos are easily removed, however, from the saved images by procedures of image enhancement and adjustment of grey-level thresholds, and they caused no problem in subsequent image analysis. 2.3. Epijkorescence integrator microscopy, video camera and

2. Materials and methods 2.1. Cultures Cultures of cyanobacteria were grown in incubators at 20C under a photon irradiance of 20 pm01 me2 s-. Filament length analyses were made of the undifferentiated filaments of Oscillutoriu rubescens BC 9303 and the heterocystous filaments

Membrane filters containing cyanobacterial filaments were observed under epifluorescent illumination with a Leitz Orthoplan microscope using a 2.5X objective. The filaments were illuminated through a Ploemopak M2 filter block, which allowed an excitation beam of wavelength 546 nm through the band pass filter and a fluorescent beam of wavelength >580 nm through the reflection short pass filter. A Cohu high performance CCD camera mounted on the camera column of the microscope was connected via a Synoptics integration circuit to a Dell 486 PC with 8 MB RAM (66 Mhz 486 DX2 CPU) and 430 Mb hard drive. The 768 X 512 pixel 256 grey level images were displayed on a section of a Sony SVGA 1024 X 768 pixel 17 monitor. The brightness of the image was enhanced using the integrator until filaments appeared as white lines against a dark ground (Fig. 1). The image was captured with a Synoptics (Cambridge Science Park) Synapse Grabber, version 1.3, and stored as a 0.39

A.E. Walsby, A. Avery I Journal of Microbiological Methods26 (1996) 11-20

Fig. 1. Micrograph

of a region of a filter sh owing the distribution

of fluorescent

filaments.

Mbyte BMP (bitmap) computer file. Images were obtained from at li:ast 5 different fields across the diameter of each membrane filter. 2.4. Analysing the images 2.4.1. Measuring total filament length The recalled images were subsequently analysed with PC-Image for VGA and Windows software, version 1.3 (Foster Findlay Associates, Newcastle). The images were edited and analysed with steps l-7 of the following procedure using program commands from pull-down menus. (Similar commands are used other image analysis software).

The image was inverted to display the fluorescent filaments as dark lines against a light background
(Process; Look up tables; Invert).

3. The images of filaments, comprising pixels within certain grey-level limits, were selected and highlighted in blue (Process; Threshold, maximum and minimum limits selected). 4. The highlighted lines over the filaments were thinned to single rows of pixels (Binaries; Morphological; Skeleton) and then thickened to 3pixel widths to render them more visible for subsequent editing (Morphological; Thicken). 5. The highlighting on spurious objects was removed (Binaries; Binary editor; Erase). Regions of filaments below the threshold were highlighted by drawing with the cursor (Binary Editor; Draw). Where filaments overlapped, additional lines were drawn parallel to the overlap. 6. The highlighted lines over the filaments were again thinned to single rows of pixels (Binaries;
Morphological; Skeleton).

The contrast of the image was enhanced (Process; Convolutions; Sharpen; Gaussian smoothing).

7. The total length, in pixels, of each of the highlighted filament images was measured and then summed (Measures; Measure all). The PC-Image

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A.E. Walsby, A. Avery I Journal of Microbiological Methods 26 (1996) II-20

software calculates several parameters for each separate highlighted object; for our procedure Detected area and Perimeter were selected (Measures; Data Analysis; Classify Objects, Statistics on Data; select Detected area, Perimeter). Once established, the above procedures were automated by recording them to the Windows 3.1 Macro recorder; the program then operated through the 7 consecutive steps with pauses only after step 3 (to adjust the threshold) and step 5 (to edit the image). 2.4.2. Enumerating and measuring lengths of individual jilaments The numbers and dimensions (Detected areas, Perimeters, etc.) of individual filaments can be retrieved from the data file established at the last step (7) of the image analysis procedure, and copied to a Microsoft Excel spreadsheet, for data analysis, but it is important for these purposes to modify the procedure in the following way.

square; in our system the average dimensions represented p, = 2.60 pm and p,, = 2.65 pm (a difference of <2%) in images from the 2.5X objective. These two calibrations can be entered as constants into the image analysis program so that results of subsequent calculations are displayed in the corresponding length units.

3. Results and discussion 3.1. Images of wet-mounted and dried jilaments

1. A microscope objective should be chosen that gives a field of view exceeding the length of the longest filaments. 2. Low concentrations should be used to reduce the number of touching filaments. 3. The highlighting on filaments contacting the edge of the screen should be eliminated (Measures; Remove edge objects) after step 3 above. 4. The highlighting on touching filaments (treated as single objects) should be eliminated (step 5 above) and the length of the remaining separate filaments analysed (step 7). 5. The analysis should then be repeated, making individual measurements (Measures; Measure object) on each of the touching filaments after erasing connections to neighbouring filaments. 2.5. Calibrating the images

The dimensions represented by a pixel were determined by calibration with the images of a stage micrometer, oriented parallel to the horizontal (X) and vertical (Y) axes of the screen, using the same microscope objective. Pixels are approximately

Living cyanobacterial filaments, mounted in water on slides, and dried filaments, collected on membrane filters, fluoresced red (or orange) against a dark ground and could be enumerated and measured directly with the image analysis system. Wetmounted filaments provided images with greater detail, which may permit different species to be distinguished in subsequent analysis. There were several advantages, however, in performing the analyses on filaments on membrane filters. The filaments did not move and they all lay in the same focal plane. The fluorescence of the filaments increased as the membrane filters dried, giving greater sensitivity. The fluorescence remained stable in samples stored in air at room temperature for 2 years or more; the samples could be reanalysed. The fluorescence of the dried filaments faded much less than that of the wet-mounted ones under prolonged exposure to the high intensity excitation beam. The required filament concentration could be produced by controlling the volume of sample filtered. Video images of Anabaena flus-aquae filaments taken with the 10X objective gave fragmented lines owing to the constricted connections between cells; at a lower magnification, with a 2.5X objective, the lines were continuous except where heterocysts of low fluorescent intensity occurred. Undifferentiated filaments of Oscillatoria spp. gave continuous lines; filaments that contained the orange-fluorescing pigment phycoerythrin gave brighter images than filaments that contained only the red-fluorescing pigment phycocyanin. In analysing lakewater samples it was found that the cells of green algae, diatoms, dinoflagellates and other algae fluoresced much less strongly than

A.E. Walsby, A. Avery / Journal of Microbiological

Methods 26 (1996)

11-20

15

cyanobacteria; their images were eliminated from the computer image by setting the upper grey-level threshold at an intermediate value (usually between 90 and 160, depending on the image brightness). 3.2. Determining filament length

square of side p, the area of a straight line of n pixels oriented either in a row on the X axis or in a column on the Y axis is rips, and the length is 1 = np,. The length of a line oriented at an angle, 19, between 0 and n/4 radians (4.5) to the X or Y axis, is 1, = np,lcos0 (1)

The length of a filament can be calculated from the highlighted array of pixels (the Object) in the image. The PC-Image software contains facilities that will calculate a number of parameters from the number and arrangement of the pixels, but none that will calculate length directly. (A facility named Length is inappropriate as it measures the maximum chord length, e.g. the major axis of an ellipse or a C-shaped line). We describe the use of Detected area and Perimeter for measuring filament length, and complications that arise in each case. 3.3. Detected area method The Skeleton function in Binary Editor is used to erode the highlighmd band of pixels over a filament to a single unbroken line of pixels. If each pixel is a

The value of 1 /cos6 varies from 1.0 (at 13 = 0) to d2 = 1.414 (at 8 = r/4). This error is illustrated in Fig. 2, which shows the lengths of straight lines of 1 pixel width drawn (by using the facility Binary editor, Draw segment) at different angles across the image of a 360 protractor. The measured pixel areas of these lines showed a minimum at angles of 7r/4 (45) from the X or Y axes; multiplying the pixel areas by 1 lcos8 gave corrected line lengths that were all within 2% of the mean diameter of the circle (Fig. 2). (An additional minor correction for pixel asymmetry, described below, is also included in Fig. 2). For a large array of lines oriented at all angles the average correction is therefore obtained by integration,

450

50

c
15 30 45 60 75 90 105 120 135 150 165 180

Angle I degrees
Fig. 2. Estimates of the length of images of lines of approximately the same length drawn at different angles (0) and skeletonised to files of 1 pixel width from measurements made by: (m) Perimeterl2; (A) Detected area/p; and (0) Detected area/p co& (see text for precise details).

16
9714 r =

A.E. Walsby, A. Avery I Journal of Microbiological

Methods 26 (1996) 11-20

(4/%-)

1 /cost d0 = 1.1222 (2)

= (4/r)

;-lntan(7r/X),

This factor of 1.1222 can be used to provide an average correction term for randomly oriented arrays of filaments. In theory applying this correction could cause errors of between + 0.1222 (all lines horizontal (all lines at 45). In or vertical) and -0.2920 practice, the lines are usually oriented at a range of angles and the errors are much less (see Fig. 3). 3.3.1. A minor correction for pixel asymmetry Because the pixels are not exactly square, the length of a straight line of n pixels oriented in a row on the X axis is I, = np,, and that in a column on the Y axis is 1, = np,. A straight line of n pixels at an angle 13between 0 and tan-(p,/p,) (= 45.5 in our system) to the X axis crosses n columns of pixels and therefore has a length of np,lcostY; a line at greater angles (between 45.5 and 90) has a length of np,lsint? These additional corrections were used in calculating the line lengths from measurements of Detected area in Fig. 2.

This type of correction cannot be automated for making measurements on an array of lines by the Detected area method. Measurements are therefore based on the geometric mean value, p = (pxpy)o.5, which in our system would generate an error with a maximum of 1% (all lines vertical and/or horizontal) and usually much less. In summary, to calculate the total line length, the total detected area of the skeletonised objects is divided by the geometric mean of p, and pY and multiplied by 1.1222. Corrections for overlapping lines are discussed below. 3.4. Perimeter method

y=o.976& Rz=0.998

old
0
locm 2oc4 3GQo 4cm
I

5ooo pixels

6ocu

7!m

detected area x 1.1222

Fig. 3. Comparison of measurements made of the length of Oscillatoria filaments by Perimeterl2, corrected by addition of 0.619 for each filament end, and Detected area/p (multiplied by 1.1222, the average value of 1 /cost?, to correct for angle of orientation).

An alternative method of determining total filament length is to use the facility Perimeter, which determines the length of the edge of a highlighted object. The software in PC-Image calculates the length of a smoothed line along the edge. Measurements made on diameters drawn on a circle at different angles indicate that the mean error of the calculation is only 0.65% for lines over all angles, with the greatest error being 1.1% for lines at angles of 10 from the vertical or horizontal axes (see Fig. 2). The length of the skeletonised line is half the perimeter; the measurement needs no additional correction for orientation and in this respect is preferable to the Detected area method. (In systems that do not provide this smoothing facility a correction must be applied; see below). The Perimeter method incurs a problem, however, when crossing filaments enclose islands of space: the pixel edges bordering the enclosed space are not measured. The solution is to erase a highlighted pixel in one of the bordering lines. The number of pixels lost in the erasures (given by the difference in Detected area before and after the erasures) should be noted and added to the final perimeter value. In practice there is an upper limit to the maximum filament concentration that can be analysed by the Perimeter method. It is difficult to keep track of more than 30 erasures and the smaller enclosed spaces that occur at high concentrations are easily overlooked. We investigated the relationship between the number of enclosed spaces and total filament length: the numbers were negligible at concentrations below 3000 pixels (= 9 mm) per image, but rose

A.E. Walsby, A. Avery I Journal of Microbiological Methods 26 (1996) 11-20

17

400

300

200

100

0
0

5000

10000

15000

20000

25000

total filament length / pixels

Fig. 4. The relation between number of filament (line) intersections (m) and the number of islands of space isolated by filaments (0) in relation to the total filament length (number of pixels) on the screen. The formula of the line for the curve (n = n + [nl 1005].75 + [nl 10 000]5 ) can be used to correct for the pixels counted only once at the intersections.

steeply to about 60 at total filament lengths of 15 000 pixels (= 45 mm), and to 300 at lengths of 25 000 pixels (= 75 mm) (Fig. 4). 3.4.1. An average correction for unsmoothed Perimeter methods In some image analysis systems the length of pixels at the edge of an object may be measured either as length p or pd2/2, depending on whether they contact the edge or the corner of the next adjacent pixel. These give exact lengths of lines that are horizontal, vertical or at 45 (or, more precisely, at tan- (p,/p,), ,45.5 in our system), but give overestimates at other angles. Consider an image of a straight line of length z at an angle 8 to the horizontal rows of pixels. The skeletonised file of pixels will cross y rows and x columns of pixels in which $ =

The skeletonised line will contain y pixels that make comer contacts to pixels in the next row and (x - y) pixels that make straight contacts. Ignoring the end pixels, the total length of the line would therefore be calculated by Perimeter12 as I = yV2 Substituting + (X y)l = x + y(V2 1) (5)

from Eq. (4), the calculated 1)

length is (6)

1 = x + Man&2

while the true length, from Eq. (3) and Eq. (4), is z = (x2 + (xtanf9)2)05 Putting x = 1, the relative difference I and z is calculated as A = (2 -2)/z = [l + tan@2 + tan26))0.5 1) (1 + tan20)0.5]l(1 (8) (7) (A) between

x2 +

y2

(3) (4)

y = xtant?

The value of A rises from 0 (at 0) to a maximum

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A.E. Walsby, A. Aveq

I Journal of Microbiological

Methods 26 (1996) I1 -20

of 0.0824 at r/8 radians (22.5) and then falls back to 0 at 7~14 (45). The user is advised to check the system by comparing the length of images of a scale line (e.g. a stage graticule) oriented at different angles by the perimeter method. If the line at 22.5 is about 8% longer, then a correction should be applied. The average overestimate for lines at all angles is given by integration of Eq. (8) between angles of 0 and ~14 (45).
7rl4

In summary, the Detected area method is preferable to the Perimeter method as it requires no editing of bordering lines and is unaffected by errors associated with this at higher filament concentrations. 3.6. Crossing, overlapping and touching jilaments

i
0 -

{[1 + tan&2

1) + tan28)0.5}dB (9)

(1 + tan20)0-5]l(1

= 0.0548

In such systems the values given by Perimeter12 should therefore be corrected by multiplying by l/ 1.0548 = 0.948. 3.5. Comparison of the Detected area and Perimeter methods For straight lines of 1 pixel width drawn parallel to either the X or Y axes the measurement given by the Perimeter-12 was always 1.382 pixels less than the measurement given by Detected area, i.e. the terminal pixels are each measured as (2 - 1.382) = 0.618 pixels by Perimeter. For straight lines drawn at other angles we found that the measurement given by Perimeter-12 agreed closely with that given by Detected area/co@ when the small correction described above for the difference in p, and py was applied (see Fig. 2). We also compared measurements, by the two methods, of filament lengths from 20 images of Oscillatoria rubescens filaments in which the total filament length ranged between 150 and 7000 pixels (= 0.4 and 18 mm). The measurement of Perimeter/ 2 was corrected by addition of 1.382012 where D is the number of ends; Detected area was multiplied by 1.1222, the correction for angle. The Detected area method gave measurements that were on average 2.4% higher than the Perimeter method (Fig. 3). At higher filament concentrations the discrepancy increased, probably due to the difficulty in locating enclosed spaces.

If two filaments intersect, the total length of the skeletonised lines is underestimated by I pixel. The proportion of intersections rises with the line density (in theory as a function of n2). An analysis of 33 fields, containing skeletonised images of Oscillatoria rubescens filaments, indicated that the error rose from <O. 1% when the detected area was < 1000 pixels, to 0.6% at 12 000, and 1.7% at 26 000 pixels per screen (of 393 216 pixels). To avoid laborious counting of the number of intersections on each image, a general correction to the total filament length can be obtained from the formula of the curve fitted to the data in Fig. 4: n = n + (n/1005)75 + (n/10 000)53 (10)

The regions where filaments overlap or lie adjacent to one another are easily distinguished as lines of greater brightness (darkness after inverting), often between branches. Such regions generate only a single line of pixels when the highlighted lines are skeletonised. To correct for this it is necessary to draw an additional line of pixels parallel to and the same length as the region of contact. The time taken to edit the image in this way rises with filament concentration in a similar manner to that for making erasures in island borders. These corrections should be applied to the lengths determined by both the Detected area and the Perimeter methods. 3.7. Sampling errors

The image analysis system provides an accurate and rapid method of determining total filament length in a field of view. With this method it is feasible to obtain much larger data sets than by previous methods. In the course of a study on seasonal changes in the population of Oscillatoria rubescens in Lake Zurich we have analysed many images of the filamentous

A.E. Walsby, A. Avery I Journal of Microbiological Methods 26 (1996) 11-20

19

80

y = 680 ho,46

5000

10000

15000

20000

25000

30000

mean number of pixels per image Fig. 5. The relationship between the standard deviation and total filament length per image. Each of the 141 points indicates the standard
deviation of measurements of images from 5 different areas of the same filter. The area analysed is 391 937 pixels (= 2.7 mm).

cyanobacteria collected on filters (A.E. Walsby and F. Schanz, in preparation). Fig. 5 shows the variation in standard deviation of the mean of 5 images from each sample, related to the mean filament length per image. As expected, the standard error trend decreases as a hyperbolic function of filament concentration. This data set draws attention to the large standard errors associated with measurements on distributions of filaments. For a random distribution of lines the standard error depends on both the number and length distribution of the line.

References HI Olson, F.C.W. (1950) Quantitative

estimates of tilamentous algae. Trans. Am. Microsc. Sot. 59, 272-279. [21 Gibson, C.E. (1975) Cyclomorphosis in natural populations of Oscillaforia redekei Van Goor. Freshwater Biol. 5, 279286. I31 Booker, M.J. and Walsby, A.E. (1981) Bloom formation and stratification by a planktonic blue-green alga in an experimental water column. Br. Phycol. J. 16, 411-421. [41 Oliver, R.L. and Walsby, A.E. (1984) Direct evidence for the role of light-mediated gas vesicle collapse in the buoyancy regulation of Anabaenu jos-aquae (cyanobacteria). Limnol. Oceanogr. 29, 879-886. VI Konopka, A. (1982) Buoyancy regulation and vertical migration by Oscillatoria rubescens in Crooked Lake, Indiana. Br. Phycol. J. 17, 427-442. t61 Konopka, A., Brock, T.D. and Walsby, A.E. (1978) Buoyancy regulation by planktonic blue-green algae in Lake Mendota, Wisconsin. Arch. Hydrobiol. 83, 524-537. [71 Bott, T.L. and Brock, T.D. (1970) Growth and metabolism of periphytic bacteria: methodology. Limnol. Oceanogr. 15, 333-342. Is1 Bailey-Watts, A.E. and Kirika, A. (1981) The assessment of size variation in Loch Leven phytoplankton: methodology and some of its uses in the study of factors influencing size. J. Plankton Res. 3. 261-282.

Acknowledgments We wish to thank Dr. F. Schanz, University of Zurich, for providing filtered samples for analysis, Mr Tim Colbum for assisting with drawing image templates and photography, and Professor B. Silverman for discussion of statistical methods. This work was supported by grants GR9/1201 and GR3/8018 from the Natural Environment Research Council.

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A.E. Walsby, A. Avery I Journal of Microbiological

Methods 26 (1996) II-20

191 Sheath, R.G., Morison, M.O., Cole, K.M. and Valanstyne, K.L. (1986) A new species of freshwater Rhodophyta, Bactrachospermum carpocontortum. Phycologia 25, 321330. [IO] Sheath, R.G. and Cole, K.M. (1990) Bactrachospermum heterocorticum sp. nov. and Polysiphonia subtilissima (Rhodophyta) from Florida spring-fed streams. J. Phycol. 26, 563-568. [ 1 l] Hoogveld, H.L. and Moed, J.R. (1993) A digitising tablet for determining the length distribution of filamentous cyanobacteria. Eur. J. Phycol. 28, 59-61.

[12] Brock, T.D. (1978) Use of fluorescence microscopy for quantifying phytoplankton, especially filamentous blue-green algae. Limnol. Oceanogr. 23, 158-160. [ 131 Daley, R.J. and Hobbie. J.E. (1975) Direct counts of aquatic bacteria by a modified epifluorescence technique. Limnol. Oceanogr. 20, 875-882. [14] Waterbury, J.B., Watson, SW., Guillard, R.R.L. and Brand, L.E. (1979) Widespread occurrence of a unicellular marine planktonic cyanobacterium. Nature (Land.) 277, 293-294.