Journal of Applied Phycology 14: 193–204, 2002.

193
© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

Cell identification and sizing using digital image analysis for estimation of
cell biomass in High Rate Algal Ponds

A.J. Gray 1,*, D. Young 1, N.J. Martin 2 and C.A. Glasbey 3

1
Department of Statistics and Modelling Science, University of Strathclyde, Livingstone Tower, 26 Richmond
Street, Glasgow, G1 1XH, UK; 2formerly Biochemical Sciences Department, Scottish Agricultural College,
Auchincruive, Ayr, KA6 5HW, UK; 3Biomathematics and Statistics Scotland, JCMB, The King’s Buildings,
Edinburgh, EH9 3JZ, UK; *Author for correspondence (e-mail: alison@stams.strath.ac.uk)
Received 14 July 2001; accepted in revised form 14 January 2002

Key words: Algae, Biomass estimation, Cell, Differential interference contrast (DIC), High rate algal ponds, Im-
age analysis

Abstract

Current environmental concerns make estimation of microbial biomass a priority for monitoring purposes and to
advance scientific understanding. This paper considers problems associated with algal cell imaging and measure-
ment for cell biomass estimation in samples from high rate algal ponds. In a complex system, the only way of
measuring microbial activity is to measure the individual cells and estimate biovolumes. Accurate biomass de-
terminations demand direct microscopic counting and measurement of the sizes of individual microbial cells taken
from known volumes of water. The system used for routine measurement at the laboratory where the images
were generated, based on standard microscope equipment, is only suitable for treatment of well dispersed speci-
mens. Differential interference contrast (DIC) microscopy, on the other hand, offers the best solution for optical
enhancement of cell contrast, and produces an image with well defined edges, yet presents a great challenge to
routine cell identification by digital image analysis, owing to the bas-relief type image produced. The paper out-
lines several image analysis methods developed specifically for this purpose, and presents illustrative results.

Introduction the whole population or fractions of the population.

In microbiology the estimation of microbial biomass
is a long standing problem. The work discussed in
this paper arose from a need to estimate microbial
biomass in high rate algal ponds (HRAPs) (Fallow-
field and Martin 1990; Cromar et al. 1996), for treat-
ment of agricultural, industrial or human waste. Op-
timising efficiency of these systems for
implementation in different climatic zones requires
understanding of the factors controlling photosynthe-
sis in the pond. In turn, this demands knowledge of
the biomasses of the photosynthetic algae and of the
non-photosynthetic bacteria and other organisms that
comprise the active biomass. Many routine methods
for biomass estimation exist, either indirect methods
which measure key components such as chlorophyll,
or direct measurements of the dry matter content of
Typical problems with such measurements are that
cells can vary in their unit chlorophyll content, the
biological composition of population fractions can
vary, or that dead cells and organic debris may be in-
cluded in dry weight measurements. These quick
techniques also need calibration by direct measure-
ments of biomass. Direct microscopic counts of algal
numbers avoid these problems, but do not give any
estimate of biomass. For biomass, information is
needed on cell size as well as number. Traditionally,
the only methods suitable for complex mixed popu-
lations of cells have involved laborious manual mea-
surement of cells.
The biomass of algae within a pond can be esti-
mated from a sample of pond water of known vol-
ume. After preparation to ensure as uniform as possi-
ble a dispersion of the cells and their presentation as
194
a film with a defined thickness, and therefore sample
volume per unit area, the sample can be examined
under a microscope and counts or measurements then
be made from the resulting microscope image. In par-
ticular, visualisation of cells by microscope imaging
makes it possible to distinguish between cell types,
and hence separate measurements for each cell type
to be taken. Biomass can be estimated by measuring
the projected (i.e. on screen) area and perimeter, or
dimensions, of each single cell and using standard
formulae (Jenkinson et al. 1976; Young et al. 1998),
based on cell shape or approximations to cell shape,
to convert to an estimate of biovolume per unit vol-
ume of pond water. Using direct measurements of cell
density (Cromar et al. 1996), or literature values, the
biovolumes can be converted into biomasses from
which total pond biomass can be estimated. In order
to obtain statistically valid measurements, a standard
requirement in bacteriology is that at least 300 cells
per sample need to be examined. It is clear that man-
ually measuring cell size becomes a monumental task.
An automatic (or even semi-automatic) method for
analysing algal cell images, localising cells and tak-
ing required measurements, is a more practical alter-
native for routine analysis.
Our objective is to develop methods for estimat-
ing cell numbers and volumes in mixed and aggre-
gated microbial populations. The ultimate aim is to
develop fully automated methods for biomass estima-
tion from microscope images, though the initial ob-
jective is to produce simpler tools to aid counting
cells. This paper summarizes difficulties in the digital
analysis of algal cell images and discusses suitable
microscopy, before reviewing recent developments
and approaches to the image analysis. Finally illus-
trative results are presented to show what can cur-
rently be achieved.
Problems in cell visualisation and identification
Unfortunately, there are many difficulties involved in
computer recognition of cells in a complex mixed
population. In a natural population, cells tend to be
clumped, with overlapping cells often of different
sizes. Preparations are rarely confined to a single
plane and there are usually out-of-focus cells.
Many digital segmentation algorithms are severely
limited by their inability to cope with the complexi-
ties of the images to be considered, namely the need
to treat contiguous and overlapping cells as individual
units, presence of many different types, shapes and
sizes of cells, detritus material, blurring from out-of-
focus cells, and non-uniformity of cytopasmic density
and/or stainability within many cells. The major prob-
lem is lack of sufficient image contrast together with
clumped cells.
For the computer to identify individual cells, there
must be sufficient contrast between the cells and their
surroundings. This can be provided by staining, but
this is not always suitable for analysis of living cell
preparations; also the best staining technique, fluores-
cent staining, involves very specialised instrumenta-
tion (Herman and Jacobson 1990). Optical enhance-
ment of cell visibility can be provided at low
magnifications by reducing numerical aperture of the
microscope, but this results in limited resolution and
is only suitable for well dispersed preparations.
Some successful digital cell counting methods, for
microscope images of cell samples, have been devel-
oped, and some commercial systems are available.
However these tend to use dedicated hardware and
proprietry software. Brown et al. (1989) reported an
automated algorithm, using readily available hard-
ware, for counting, sizing and morphological descrip-
tion of algal cells imaged by brightfield microscopy,
based on histogramming and thresholding. Bjørnsen
(1986) and Psenner (1991) addressed detection, siz-
ing and biomass estimation of aquatic bacteria for
epifluorescence microscopy; they used background
subtraction and filtering to reduce blur, followed by
thresholding to detect the bacteria. None of these ap-
proaches can separate touching or clumped cells typi-
cal of most microbial ecosystems, and they only work
with single microscope modalities. Garrido et al.
(1997) consider cell image segmentation in cytologi-
cal microscope images similarly complex to ours, al-
though their samples are stained. They use a Canny
edge detector with a multiscale representation of the
image, to give an elliptical approximation to the cell
outline, followed by stochastic deformation to fit lo-
cal outline irregularities. The method works reason-
ably well but would require adaptation for the DIC
images considered here (such as in Figure 1).
Materials and methods
Cell samples used
Two sources of algal cells were used for the images
in this paper; a stock culture of Chlorella vulgaris and

Figure 1. DIC image of algal cells (Chlamydomonas sp.).
naturally occurring Chlamydomonas sp. cells from a
small scale HRAP run at Auchincruive. The yeast
cells used were taken from a laboratory culture of
Candida utilis.
Image capture and analysis system
The image capture system consisted of an Olympus
Vanox microscope fitted with a Panasonic model WV-
CL700 solid state (charge coupled device, CCD)
video camera, and an Elonex 386 P.C. (and monitor)
fitted with a VP1100-KIT-512-E-C1-AT Overlay
Frame Grabber Kit image capture board (from Data
Cell Ltd., 10 West End Road, Mortimer Common,
Reading, Berkshire RG7 3SY, UK) and equipped with
a separate large monitor for image display. The pho-
totube was fitted with an Olympus FK X3.3 compen-
sating eyepiece. Once the biologist has the required
cells in view, the image can be captured and stored
digitally on computer.
The brightfield and DIC images were produced us-
ing an Olympus Plan X40 0.65NA. flat field objec-
tive, and the phase contrast image produced with an
Olympus LWDCD Plan X40 0.55NA. long working
distance objective.
Currently the laboratory scientist interacts with the
displayed image using the menu-driven P.C. software
package OPTIMAS (from Data Cell Ltd., as above).
This provides basic measurements such as area, pe-
rimeter, and cell dimensions of segmented objects,
with output going to the P.C. monitor. Automatic lo-
cation of the cells to be measured has so far meant
195
simply using thresholding to segment the cells, using
brightfield microscopy with a X10 or X40 objective.
The cells are made visible by increasing contrast by
closing the condenser iris diaphragm, thus reducing
effective numerical aperture of the objective. Un-
wanted thresholded objects can be deleted using the
editing facility and mouse-based point-and-click. The
user can also select object criteria to classify thresh-
olded objects so that only certain classes are mea-
sured, e.g. objects satisfying set size and shape crite-
ria. The biomass calculations are then done separately
using a spreadsheet package. However this procedure
only works for fully dispersed cell suspensions; also
the image quality is poor (see Figure 2a), and resolu-
tion is reduced making the method unsatisfactory for
small cells and especially for bacteria. The poor im-
age quality makes dealing with overlapping or aggre-
gated cells very difficult, hence better quality images
with well defined edges are required.
Microscopy
Optical enhancement of cell contrast can best be
achieved by phase contrast or differential interference
contrast (DIC) microscopy (Pluta 1989; Slayter and
Slayter 1992; Cogswell and Sheppard 1992; Holmes
and Levy 1987). Both techniques enhance interfer-
ence in the light transmitted through the specimen, in
order to make nearly invisible objects darker than
their background. However, phase contrast (Figure
2b) is unsuitable for measurement of cell sizes, as cell
edges are poorly defined (owing to a halo of out-of-
focus light around areas of high optical contrast), and
it gives poor images where there are overlying or un-
derlying out-of-focus cells. DIC microscopy (Figure
2c) does give visually clear images of cells with well
defined edges, and offers the best prospects for cell
recognition. However, DIC microscopy poses a great
challenge to automatic image interpretation because
of its pseudo bas-relief image, where one side of a
cell is lighter than the background and one side
darker, with a background level intensity in between.
DIC images therefore require new, specialised seg-
mentation procedures, which must also be able to deal
with the complexities described. Even if adequate op-
tical contrast can be obtained, it is still necessary to
separate individual cells.
Figure 1 shows a typical DIC image of a sample
of algal cells from an HRAP, which illustrates some
of the problems described. The cells are clustered,
and such touching and/or overlapping cells are diffi-
196
Figure 2. A Chlorella vulgaris specimen imaged by three different microscope modalities.
cult for a computer to recognise as separate objects.
The light and dark cell edges also cause difficulty, al-
though the cells are clearly distinct to the human eye.
Figures 2a, 2b and 2c illustrate three types of micro-
scope modality for another algal cell sample. Figure
2a is a brightfield image, so the cells appear as dark
objects on a lighter background. The same cells are
shown in Figure 2b imaged by phase contrast micros-
copy. Figure 2c shows a DIC image of the same spec-
imen.
Optics of DIC microscopy
In DIC microscopy, a plane-polarised beam of light
is split into two mutually coherent, orthogonally po-
larised components in a beam-splitting prism. The
two components are laterally displaced by a few
tenths of a micrometre and phase-shifted (usually by
90°) relative to each other, they pass through the spec-
imen side-by-side in the direction of displacement,
and finally are recombined using another prism near
the back focal plane of the objective. A final polaris-
ing filter converts the phase differences into ampli-
tude differences. Introducing the phase shift, and
making use of the further phase shift due to taking
different optical paths through the specimen, induces
interference between the two components of each
light ray, so that near-invisible specimens become vis-
ible. The result is that, in the absence of amplitude

changes, a DIC microscope produces an image with
intensity proportional to the rate of change of optical
thickness of a specimen in the direction of shear (lat-
eral displacement) (see e.g. Slayter and Slayter
(1992): pp. 155–158), that is proportional to the phase
gradient.
For a uniform background, a uniform intensity is
observed. If the phase shift at the object represents an
increasing optical path difference, the total phase-shift
approaches one wavelength and the object appears
darker than its surround. Conversely, passing through
an area of diminished optical path reduces total phase
shift towards zero and the object appears brighter than
the surround. Therefore intensity is less than that of
the background in regions where optical path differ-
ences decrease from right to left, above that of the
background in regions where they increase from right
to left, with a neutral point in between equal to the
background intensity. The overall effect is of ’shad-
owing’ or grey level grading across the image, as if it
were illuminated by an oblique light source. The
shadowing is not as obvious for cells which are not
fully transparent. By imaging rate of change of opti-
cal thickness, rather than absolute level of optical
path difference, the DIC microscope is capable of
forming high contrast images of very thin specimens.
Review
Little is available for fully automatic interpretation of
DIC images; indeed it has been stated that for such
complex biological images fully automatic methods
are almost always inadequate (Wu and Barba 1995).
Edge-detection is an obvious approach to cell
identification; the cell outlines found form the basis
of area measurement and conversion to biomass.
However, applying standard edge-detection algo-
rithms to DIC images, for example Prewitt’s filter
(Glasbey and Horgan (1995), pp.87–89; Gonzalez and
Woods (1992), pp. 197–201), a gradient-based edge-
detector sensitive to edges in any direction, fails. This
is due to edges being detected at the transition from
light to dark pixels within cells. The result is that cells
are split, and outlines and estimated cell sizes are too
small.
Kenyon et al. (1994) use a three-dimensional cell
modelling approach, although this does require mul-
tiple optical slices through a specimen. Edge-detec-
tion, thresholding, noise removal and smoothing steps
are used to construct three-dimensional cell bound-
197
aries, from which to estimate cell volume and surface
area. Parameters, which depend on image quality and
aspects of the cell studied, have to be chosen for the
noise suppression algorithm.
Some different approaches, which can be applied
to a single image, are now described, based on edge-
tracking and template matching, for cell localisation
and sizing. Volume can be estimated separately once
accurate outlines are found. The digital analysis meth-
ods described below all require a programmable com-
puter facility.
Automatic approaches
An integration algorithm suitable for transparent cells
only, which avoids splitting cells, is described in
Young (1997). Using the fact that the DIC image re-
presents rate of change of optical thickness of trans-
parent cells, i.e. mathematically it is a first derivative
image, the image is first integrated (summed) in the
direction of the phase gradient (light to dark edges)
to recover the image of cell optical thickness. The re-
sult is rescaled to reduce accumulation of noise, then
thresholded and smoothed to remove debris from the
background. The thresholded cells are superimposed
on the original image and grown out to match better
the actual cell outlines; their boundaries are then eas-
ily found by edge-tracking. A simple procedure is
used to identify outlines of two or more touching cells
and split them.
A more generally applicable edge-tracker, which
does not require pre-processing of the image, and is
applied to the grey-level image, sequentially gener-
ates an outline by minimising a cost function using
the difference in intensity between pairs of adjacent
pixels. This is based on the approach of Martelli
(1976) for noisy images. Modification of the cost
function avoids tracing inside cells along the phase
gradient ’edge’, and a curvature constraint ensures
that the outline is completed for each single cell so
touching cells are successfully separated. Details are
given in Young and Gray (1996).
Computer-aided edge-tracking
This fully automatic procedure fails when a weak
boundary is encountered, or where artefacts with
strong boundaries occur. There are no distinct bound-
aries at some parts of the cell edges, as well as there
being more obvious edges within cells. It is therefore
198
Figure 3. Application of integration approach.
necessary to use heuristic information to enforce the
required curvature, or to interpolate the contour.
In the absence of a completely successful fully au-
tomatic procedure, a semi-automatic approach giving
accurate, reliable and quick results is still preferable
to the current fully-manual alternative. Computer-
aided or semi-automatic systems attempt to improve
on a manual interpretation system by allowing a hu-
man operator and computer to interact, thus speeding
up the process and reducing user fatigue. These are
often a compromise when a fully automatic system is
not possible due to the complexity of the problem.
Such methods are often used in practice (Wu and
Barba (1995) and Yamashita et al. (1993), Carothers
et al. (1994); and in a different context, Altman et al.
(1981)).
Young (1997) involves an operator choosing two
thresholds to segment the light and dark cell edges,
then identifying two points on the edge of each cell
of interest-one on the light side and the other on the
dark edge. The edge-tracking algorithm then traces
outlines of the cells along each of these edges sepa-
rately. (It traces in both a clockwise and anti-clock-
wise direction until the maximum cost becomes less
than a specified level, then stops, when the part of the
cell boundary reached closely matches the back-

Figure 4. Application of edge-tracking.
ground intensity). The end-points of the two contours
are joined by straight lines. For irregularly-shaped
cells this leads to slicing off parts of cell, and the
end-points are then joined by curves (by making a cell
shape assumption). Uneven illumination and less
good focus also cause some cell outlines to be less
than accurately traced. The user can choose a point
near the centre of the straight line and ’pull’ it down
to better represent the true cell outline. The improve-
ment in accuracy is at the expense of the user identi-
fying further points for adjustment. The accuracy of
this simple method can also be improved if the op-
erator identifies any points where touching cells have
not been split. Further operator input at this step ad-
justs contours as required to give more accurate out-
lines.
Template matching
Template matching involves sliding a sub-image or
template across an image and finding locations in the
image where the sub-image matches most closely; it
is most applicable for identifying cells which are sim-
ilar in shape. A model cell can be used as a template
to identify the location of cells. Good matches corre-
spond to positions with optimum values of a speci-
fied criterion of goodness-of-match between the tem-
plate and the area of overlap with the image. Once
cell positions are identified, estimates of cell size can
be obtained by matching different sizes of the tem-
199
plate fitted to the identified cell centre. Template
matching is rarely used in practice as a method of
image analysis, due to its computational intensity,
however use of Fast Fourier Transforms (Gonzalez
and Woods (1992): Ch. 3.4) vastly reduces process-
ing time and does make it practically feasible if a
suitable matching criterion is chosen.
Young et al. (1998) and Young and Gray (1998)
give details of a template matching procedure to suc-
cessfully locate and size cells. A key step is construct-
ing an idealised model cell for use as a template; one
which closely resembles an actual cell in the image.
Different template construction methods are appropri-
ate for different types of cell, i.e. transparent and non-
transparent cells, cells with shapes capable of geomet-
ric description (spherical and ellipsoidal), and cells
with less easily described shapes. This is most com-
plex for cells which are not fully transparent. A mod-
elling approach is used, using a mathematical repre-
sentation of the DIC image of the cell, based on
theoretical properties of the microscope optics.
It can be shown that at location (i, j) in the image
plane the DIC image grey level f(i,j) can be repre-
sented as f(i,j) ⬇ b(i,j)×d(i,j), where d
d dt
共i, j兲 ⫽ 1 ⫹ k t共i, j兲, and is the directional deriv-
d␯ d␯
ative of optical thickness in the direction of shear ␯,
estimated by image differencing; k represents refrac-
tive index and kt(i,j) is optical thickness. So f(i,j) ⬇
200
Figure 5. Computer-aided approach (for Chlamydomonas).
d(i,j) if the specimen is transparent. The term b(i,j) is
the corresponding brightfield representation, which
may be taken as b(i,j) = a 2e −ct(i,j), where a and c are
non-negative constants (light wave amplitude and rate
of light absorption), if constant optical density of the
specimen is assumed. A subsequent baseline shift l in
grey levels by the digitising camera may be allowed
for, as may blur by convolution of f(i,j) with a Gaus-
sian blur function with standard deviation s, for ex-
ample. This is used to represent blur from a number
of sources (the p.s.f. of the optical system, blur in-
duced by the digitising camera, and out-of-focus parts
of the image); for detailed study of the actual form of
blur due to the optical point spread function see Preza
et al. (1996).
For cells of specifiable geometric shape, the cell
template grey levels are modelled; for example for an
ellipsoidal cell, geometric thickness at the vertical
axis passing through (i,j) in the image plane of an el-
lipsoid centred on (0, 0) and with semi-axis lengths
␴ i and ␴ j is proportional to t(i,j) = 2{1-[(i/␴ i) 2 + (j/
␴ j) 2]} 0.5 if this is defined, otherwise t(i,j) = 0. Assum-
ing constant refractive index of the cell, the result is
proportional to optical thickness. The resulting tem-
plate cell is rotated as necessary before first order
differencing in the appropriate direction.

Figure 6. Further computer-aided results (Chlorella).
Parameters in the model are estimated to minimise
discrepancy between the model cell grey levels and a
cell extracted from the image, using the least squares
method. A range of templates at different rotations are
modelled for use in matching, using the parameter
estimates, as well as matching with a range of differ-
ent cell sizes. This means rotating the brightfield com-
ponent {b(i,j)} and the cell thickness template {t(i,j)}
separately before multiplying and blurring.
201
Results
First we illustrate the inadequacy of a simple edge
filter, then show results for the DIC-specific ap-
proaches described above.
Edge-filtering
Prewitt’s filter applied to Figure 2c produces the re-
sult in Figure 2d. There are double boundaries, edges
within cells and considerable noise.
202
Figure 7. (Chlorella) Template matching result.
Automatic approaches
The integration algorithm for transparent cells is il-
lustrated on Candida yeast in Figure 3. Figure 4
shows a sample of Chlorella and final results of edge-
tracking using the cost function approach.
Computer-aided edge-tracking
Results for Figure 1 are shown in Figure 5. Two dif-
ferent Chlorella images are shown in Figure 6.
Template matching
Figure 7 shows marked cell centres and outlines for
Figure 4a, using the template matching method for
non-transparent cells. The cells are assumed to be
spherical. The cell modelling is illustrated in Figure 8,
which shows an extracted image cell (Figure 8a), the
corresponding modelled cell (Figure 8b) and residu-
als between the two (Figure 8c) (zero is shown as
mid-grey, and the residuals appear acceptably ran-
dom). Very slight improvement in fit is possible if el-
lipsoidal outlines are fitted. The template method for
non-transparent, non-spherical cells gives the result in
Figure 9. Cell outlines are generated by the best fit-
ting template matching the cell.
Discussion
Various methods have been considered for digital cell
identification in DIC images.
Simpler automatic approaches
The integration approach is only applicable for fully
transparent cells. The edge-tracking method is more
general, and for the fairly distinct cells in Figure 4 it
is fast and accurate. However, neither method is ideal
for heavily clustered cells and user-input is required
for more generally acceptable results.
Computer-aided edge-tracking
This method is flexible in that the amount of user-in-
tervention can be tailored to balance required accu-
racy of results and available operator time. In Fig-
ure 5, for example, further improvement is clearly
still possible.
Although it is very simple and fast, this method
leads to an irregular cell boundary. A smoother result
could be achieved by finding the best fitting ellipse,
say, fitting the set of points comprising the irregular
boundary. This then allows an estimate of cell volume
from standard formulae (see Jenkinson et al. (1976)
and Young et al. (1998)).
Young and Gray (1997) consider several other
computer-aided techniques making assumptions of
cell shape, where shape can be specified. These have
the advantage that less user-input is needed; a region-
growing algorithm, for example, simply requires the
operator to identify a single point on each cell.
Template matching
The results shown use the three-step matching proce-
dure of Young and Gray (1998) to locate and mask
out larger cells, medium-sized then smaller cells in
turn. Young et al. (1998) describe an alternative single
step method using a modified template (in which pix-
els in a narrow border around the template cell were
set to black or white, the value of adjacent pixels at
that part of the cell if it were touching a neighbour-
ing cell).
Further speed-up of computer time could be
achieved by a semi-automatic procedure, where the
user supplies a seed point for each cell to identify an
approximate cell centre, so matching to locate cells
takes place only over a small area around each seed

Figure 8. (Chlorella) Cell modelling.
Figure 9. (Chlamydomonas) Template matching result.
point rather than the whole image; alternatively hier-
archical matching could be used, in which matches
are found for a coarsened image containing fewer pix-
els (and so fewer possible cell centres) and then re-
fined for successively less coarse images until the
outlines are fitted to the original image.
An operational system
The template matching, edge-detection and semi-au-
tomatic methods presented for identification and siz-
ing of cells in DIC images provide building blocks
from which an operational system for cell biomass
estimation may be developed for use in applications
requiring cell counting. The computer-aided approach
may be the most promising one to explore further to
203
develop a working system for routine analysis of
HRAP water samples.
Important problems remaining include classifica-
tion of different cell types in a mixed cell sample, and
dealing more effectively with occluded or overlap-
ping cells. High level Bayesian statistical methods
(Baddeley and Van Lieshout 1993; Green 1995; Ri-
chardson and Green 1997; Mardia et al. 1997; Hurn
1998; Rue and Hurn 1999), though very computation-
ally intensive when the image grey level model is
complex, have the potential to cope with these, if a
statistical model can be proposed for noise in the DIC
image. Current work is investigating these methods
for DIC images.
Ideally, an automated cell image analysis system
would incorporate a computer algorithm to analyse
the images as they are captured. The biologist would
capture a field of view from the sample preparation,
which is then stored on computer. The computer
would then analyse this image, recording the number,
size and ideally biomass, of cells in that field. The
operator would then re-position the field of view, and
the process continues until sufficient fields of the sam-
ple slide have been examined. Finally, a biomass es-
timate for the cells in the pond sample would be
available. To deal with mixed cell types, a classifica-
tion tool will be a necessary part of such a system.
The template matching procedure addresses this to
some extent.
The steps in the analysis could be reorganised as
required. For instance, the biologist could capture the
images from the sample and store them on computer
for analysis later. An automated system could analyse
the images overnight, reducing time taken during the
204
working day. With the development of automated mi-
croscopes, it may become possible for a slide to be
analysed without any human input at all. A sample
slide could be mounted on the microscope stage and
the microscope would automatically scan the slide,
storing images from each sampling point. The entire
set of images obtained from a single slide could then
be analysed.
References
Altman D.G., Paton K.A., Slavin G., Taylor A.C. and Ward P. 1981.
Measurement of muscle fibre diameter by computer aided de-
sign. Int. J. Man-Mach. Stud. 14: 437–447.
Baddeley A.J. and Van Lieshout M.N.M. 1993. Stochastic geom-
etry models in high-level vision. In: Mardia K.V. and Kanji
G.K. (eds), Statistics and Images 1. Carfax, Oxford, pp. 231–
256.
Bjørnsen P.K. 1986. Automatic determination of bacterioplankton
biomass by image analysis. Appl. envir. Microbiol. 51: 1199–
1204.
Brown L.H., Gargantini I., Brown D., Atkinson H., Govindarajan
J. and Vanlerberghe G. 1989. Computer based image analysis
for the automated counting and morphological description of
microalgae in culture. J. appl. Phycol. 1: 211–225.
Carothers A., McGoogan E., Vooijs P., Bird C., Colquhoun M., Ea-
son P. et al. 1994. A collaborative trial of a semi-automatic sys-
tem for slide preparation and screening in cervical cytopathol-
ogy. Analyt. cell. Pathol. 7: 261–274.
Cogswell C.J. and Sheppard C.J.R. 1992. Confocal differential in-
terference contrast (DIC) microscopy: including a theoretical
analysis of conventional and confocal DIC imaging. J. Microsc.
165: 81–101.
Cromar N.J., Fallowfield H.J. and Martin N.J. 1996. Influence of
environmental parameters on biomass production and nutrient
removal in a high rate algal pond operated by continuous cul-
ture. Water Sci. Technol. 34: 133–140.
Fallowfield H.J. and Martin N.J. 1990. The operation, performance
and computer design of high rate algal ponds. Inst. chem. Eng.
Symp. Series 111.
Garrido A., Perez de la Blanca N. and Garcia-Silvente M. 1997.
Cell image segmentation. In: Proceedings 10 th Scandinavian
Conference on Image Analysis. Lappeenranta, Finland, pp.
659–665.
Glasbey C.A. and Horgan G.W. 1995. Image Analysis for the Bio-
logical Sciences. J. Wiley & Sons, Chichester.
Gonzalez R.C. and Woods R.E. 1992. Digital Image Processing.
Addison-Wesley, Massachusetts.
Green P.J. 1995. Reversible jump Markov chain Monte Carlo com-
putation and Bayesian model determination. Biometrika 82:
711–732.
Herman B. and Jacobson K. 1990. Optical Microscopy for Biol-
ogy. J.Wiley & Sons, New York.
Holmes T.J. and Levy W.J. 1987. Signal-processing characteristics
of differential interference contrast microscopy. Appl. Optics
26: 3929–3939.
Hurn M. 1998. Confocal fluorescence microscopy of leaf cells: an
application of Bayesian image analysis. Appl. Statist. 47: 361–
377.
Jenkinson S.D., Powlson D.S. and Wedderburn R.W.M. 1976. The
effects of biocidal treatments on metabolism in soil. III. Soil
Biol. Biochem. 8: 189–202.
Kenyon C.M., Yanai M. and Macklem P.T. 1994. Edge detection,
three-dimensional cell boundary reconstruction and volume and
surface area estimation from differential interference contrast
images. J. Microsc. 176: 152–157.
Mardia K.V., Qian W., Shah D. and de Souza K.M.A. 1997. De-
formable template recognition of multiple occluded objects.
IEEE Trans. Patt. Anal. Mach. Intell. 19: 1035–1042.
Martelli A. 1976. An application of heuristic search methods to
edge and contour detection. Commun. of the ACM 19: 73–83.
Pluta M. 1989. Advanced Light Microscopy: Specialised Methods.
Elsevier, Amsterdam.
Preza C., Snyder D.L. and Conchello J.-A. 1996. Imaging models
for three-dimensional transmitted-light DIC microscopy. Pro-
ceedings SPIE Symposium on Electronic Imaging, Science and
Technology. 2655: 245–256.
Psenner R. 1991. Detection and sizing of aquatic bacteria by means
of epifluorescence microscopy and image analysis. Im. En-
hancement Anal. 10: 13–15.
Richardson S. and Green P.J. 1997. On Bayesian analysis of mix-
tures with an unknown number of components (with discus-
sion). J. r. statist. Soc. B 59: 731–792.
Rue H. and Hurn M.A. 1999. Bayesian object identification. Bi-
ometrika 86: 649–660.
Slayter E.M. and Slayter H.S. 1992. Light and Electron Micros-
copy. Cambridge U.P., Cambridge.
Wu H.S. and Barba J. 1995. An efficient semi-automatic algorithm
for cell contour extraction. J. Microsc. 179: 270–276.
Yamashita Y., Kuwashima M., Nonaka T. and Suzuki M. 1993.
On-line measurement of cell size distribution and concentration
of yeast by image processing. J. chem. Eng., Japan 26: 615–
619.
Young D. 1997. Cell Identification in Microscope Images. PhD
Dissertation, University of Strathclyde, Glasgow, UK.
Young D., Glasbey C.A., Gray A.J. and Martin N.J. 1998. Towards
automatic cell identification in DIC microscopy. J. Microsc.
192: 186–193.
Young D. and Gray A.J. 1996. Cell identification in Differential
Interference Contrast microscope images using edge detection.
7 th British Machine Vision Conference, Edinburgh. 1: 133.
Young D. and Gray A.J. 1997. Semi-automatic boundary detection
for identification of cells in DIC microscope images. IEE 6 th
International Conference on Image Processing and its Applica-
tions, Dublin, Ireland. 1: 346–350.
Young D. and Gray A.J. 1998. Template construction and match-
ing for identification of cells in differential interference contrast
microscope images. In: IEEE 3 rd Southwest Symposium on
Image Analysis and Interpretation., Tucson, Arizona, pp. 238–
243.