JOINT DENOISING AND CONTRAST ENHANCEMENT FOR LIGHT MICROSCOPY IMAGE
SEQUENCES
Artur Loza? Mohammed Al-Mualla? Paul Verkade†
?
Khalifa University of Science, Technology and Research, United Arab Emirates
†
University of Bristol, United Kingdom
e-mail: {artur.loza, almualla}@kustar.ac.ae, {p.verkade, paul.hill, dave.bull, alin.achim}@bristol.ac.uk
ABSTRACT
This paper describes a novel method for preprocessing of mi-
croscopy images by means of denoising and contrast enhancement
in the wavelet domain. A non-linear enhancement function has been
designed based on the local dispersion of the wavelet coefficients
modelled as a bivariate Cauchy distribution. Within the same sta-
tistical framework, a simultaneous noise reduction in the image is
performed by means of a shrinkage function, thus preventing noise
amplification. The proposed method has been tested on a light mi-
croscopy image dataset and has been shown to greatly enhance the
low-contrast noisy images while outperforming other state-of-art
contrast enhancement methods.
Index Terms— light microscopy, biomedical images, wavelets,
statistical modelling, image contrast enhancement, denoising
1. INTRODUCTION
Biocellular systems perform essential processes for life, such as, for
example, the movement of material between compartments within
cells, and appropriate bioimage analysis for microscopy provides a
vital tool for medical and biochemical professionals, who rely more
and more on imaging technology to provide quantitative analysis.
Existing biological image processing tools, are often suitable only
for certain image processing tasks that often are too general in their
scope. Therefore, the real challenge lies in developing dedicated al-
gorithms for cellular bioimage processing and analysis, starting with
an accurate characterisation of data statistics. Nuclei and vesicle de-
tection in low contrast, noisy microscopy images aids greatly their
subsequent analysis. User interaction for manually achieving this
task is not an option since it is time consuming; it may be biased and
difficult to reproduce. A bioimaging pipeline needs to combine pro-
cessing methods that both improve the quality of the raw images and
enhance the information which is passed to the biologist for inter-
pretation and diagnosis. These techniques include restoration (noise
reduction and contrast enhancement) and detection/object tracking.
The former is an operation that mitigates the distortion created by
the microscope and can be addressed using various statistical ap-
proaches that model the underlying statistics using exponentially de-
caying distributions [1].
The most commonly used image Contrast Enhancement (CE)
methodologies include gray-level transformations by means of non-
linear functions, such as logarithm, power-law, gamma functions,
etc. and histogram-based techniques [2, 3]. Although the majority
of grey-level transformation methods operate in the spatial domain,
several authors have in recent years advocated the use of wavelets
and other multiscale representations for this purpose, for example
978-1-4673-1961-4/14/$31.00 ©2014 IEEE 1083
Paul Hill† David Bull† Alin Achim†
wavelets [4, 5] or steerable pyramids [6]. The advantage of using
these representations is their ability to analyse and modify image
features based on their spatial-frequency content at different res-
olutions. Early work on wavelet-based CE for medical imaging
has been reported in [4] where a parametrised hyperbolic function
was applied to the gradient of wavelet coefficients and in [5] where
echocardiographic images were both denoised and enhanced by ap-
plying a combination of sigmoid functions to high-pass coefficients.
Many other authors have reported successful enhancement with
other parametrised functions for example piecewise-linear func-
tions [7] or a hyperbolic tangent weighted by a Gaussian applied
to contourlet coefficients [8]. The multiscale-based CE techniques
have been shown to outperform other conventional techniques, such
as gamma correction and histogram stretching or equalisation in
terms of visual impression and noise suppression. However, when
using parametrised CE functions, a common issue is the choice of
suitable parameters that have to be estimated in order to compute an
optimal transfer function for a particular image. The need for auto-
mated CE procedures becomes even more obvious when processing
image sequences, where off-line estimation may not be possible.
In this work, the design of a low-complexity CE algorithm that
operates on light microscopy imagery without the need for user inter-
vention is presented. The algorithm enhances contrast in images and
image sequences adaptively, and, as opposed to global approaches,
estimates the parameters based on local statistics of the wavelet co-
efficients of an image. Moreover, the amplification of the noise in
the image is avoided by simultaneously denoising the wavelet coef-
ficients based on the same statistical assumptions.
The remaining part of this paper is organised as follows. The
proposed microscopy image enhancement method is described in
detail in Section 2. The method is evaluated on a set of microscopy
images and its performance compared with other state-of-art tech-
niques in Section 3. Finally, Section 4 presents the conclusions of
the study and suggests areas for future work.
2. IMAGE ENHANCEMENT METHOD
Generally speaking, in order to enhance an image, the low and mid-
dle intensity pixels should be boosted, while the high intensity pixels
should be either left unchanged or compressed. Special care should
be taken when boosting the small intensity pixels, so that noise in
the image is not amplified.
2.1. Theoretical Background
The evidence present in the literature and in our previous studies
(see [1] and references therein), indicates that the wavelet coeffi-
 (a) (b)

Fig. 1. Modelling of wavelet coefficients of a microscopy image: (a) empirical data vs. theoretical Gaussian data, (b) the fit of the empirical
distribution with the Cauchy, Laplace and Gaussian density functions.

cients of images frequently follow a model that deviates signifi- noise variance, σn2 , required to compute the shrinkage functions, is
cantly from the commonly used Gaussian model and can be more obtained from the observed data by computing the Median Absolute
accurately modelled by Generalised Gaussian Distribution (GGD) Deviation (MAD) of coefficients at the corresponding level of the
or Symmetric Alpha-Stable (SαS) models. A visual example of how wavelet decomposition σ̂n,j = M AD(Xj )/0.6745.
the underlying distribution of the images from our dataset deviates We begin the design of our contrast enhancement routine by
from the normal distribution is shown in Figure 1(a). The crosses defining the contrast measure. Various contrast measures have
in the plots show the empirical cumulative probability versus the been proposed in the literature, with the most common being
data values for each point in the sample. The crosses are in curves C = Lmax /Lmin or C = ∆L/Lmin (so-called Webber frac-
that do not follow the straight Gaussian lines and thus, the normality tion) where L stands for intensity (luminance) of the image pixel.
assumption is violated for all these data. Figure 1(b) compares the fit In most images defining contrast with respect to a single pixel (or,
of various density functions to the empirical distributions of wavelet in our case, a single wavelet coefficient) results frequently in an
coefficients of an image. Note that the Cauchy pdf provides a better unreliable measure, especially for high-pass image content. In [1],
fit to both the cusp and the tails of the empirical density of the actual a contrast measure has been proposed, defined in terms of the local
data. dispersion of the wavelet coefficients
In the proposed method the wavelet coefficients are denoised by
applying the shrinkage functions derived as Maximum a Posteriori γ̂(k)
C(k) = . (4)
(MAP) estimators ŵj of noisy wavelet coefficient xj corrupted by max γ̂
Gaussian white noise n: xj = wj + n, n : N (0, σn ). In particular,
for the bivariate isotropic Cauchy model (special case of SαS) used The measure (4) is calculated by means of (3) within a small 3 × 3
in this work window centred around coefficient xk,j (in the following, the level
and subband index will be omitted in the equations for simplicity)
γ
p(xj , xj+1 ) = 3 (1) and is computed with respect to the maximum dispersion in the cur-
2π(xj + x2j+1 + γ 2 ) 2
2
rent wavelet subband, max {γ̂}; its estimate serves as a reference
value towards which the wavelet coefficients are boosted in order
the following shrinkage relationship can be derived [1]: to obtain the desired contrast. The bivariate statistical model of the
xj wavelet coefficients (1), provides the basis for both denoising and
ŵj = Ad (xj )xj = + s + t, (2) contrast enhancement and accounts for interscale dependencies of
3
wavelet coefficients, assigning more importance to persistent multi-
3
q2 21 1 3
q2 21 1
where s = (− 2q + ( p27 + 4
) )3 , t = (− 2q − ( p27 + 4
) )3 scale patterns.
x2 2 2
j (γ +3σn ) x2
j 2x3 x3 2
γ +3σn 2 2 3
γ xj The local contrast values are used to adjust the corresponding
and p = x2 +x 2 − 3
, q = − 27j + 3
j
x2 +x 2 − x2 2 . wavelet coefficients by means of an exponential contrast enhance-
j j+1 j j+1 j +xj+1
In (1) and (2) γ > 0 is the dispersion parameter that determines ment function Ac
the spread of the distribution, and xj and xj+1 are the current and
parent wavelet coefficient on the level j and j + 1, respectively. The Ac (k) = e−C(k) + A0 , (5)
parameter γ is estimated based on the empirical characteristic func-
tion as follows: where A0 = 1 − e−1 is a normalisation constant ensuring that
the function converges to 1 in the areas of high dispersion. Since
log ϕ̂2 (ω) − σn2 |ω|2 shrinkage and enhancement functions are applied sequentially, the
γ̂ = − , (3)
2|ω| enhanced (i.e. denoised and contrast-enhanced) wavelet coefficient
PK is obtained as a product of the overall enhancement function and the
where ϕ̂(ω) = N1 k=1 exp(iωxk ) is the sample estimate of em- original wavelet coefficient,
pirical characteristic function ϕ̂(ω) and γ̂ is obtained as the average
of the estimates corresponding to several possible choices of ω. The ŵ(k) = Ae (k)x(k), (6)

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 (a) (b)
(e) (f)
Fig. 2. Experimental results obtained from the input image (a) with the use of the following methods: (b) denoising only, (c) CE only, (d)
proposed method, single image, (e) CLAHE, (f) Contourlet CE, (g) LAMCO, (h) proposed in 3-D (two adjacent frames).
where Ae = Ad Ah Al is the overall enhancement function, Ad is
a denoising shrinkage function (2) and Ah and Al are the enhance-
ment functions (5) of the high- and low-pass coefficients (at the cor-
responding scale), respectively.
The low-pass coefficients at the final decomposition level are op-
timised with the use of Contrast Limited Adaptive Histogram Equal-
ization (CLAHE) [2], in which the pixels overflowing the clip limit
(set to 0.01 in our work) are redistributed across the bins below the
clip limit, giving rise to a mapping function that results in the desired
shape of the histogram. Commonly used uniform distribution is suit-
able for real-world images, however, it was observed that in order
to separate the noisy background from the foreground structures in
the microscopy data, the Rayleigh distribution gives better results:
p(wa ) = wa /σr2 exp(−wa2 /2σr2 ), where wa is the approximation
coefficients and σr > 0 is the scale parameter of the distribution
1 centile back to the [0 255] range. Moreover, with the human
estimated as σ̂r ≈ (1/2N N 2
P
k=1 wa (k)) .
2
2.2. Contrast Enhancement Algorithm
The data flow of the proposed enhancement algorithm can be sum-
marised as follows:
1. RGB to HSV conversion: the RGB image is converted to Hue-
Saturation-Value (HSV) colour space and only the V-channel is
processed further. This allows reduction of the amount of pro-
cessed data and results in more consistent colouring of the output.
2. Preprocessing: the images are linearly scaled to fit the full range,
[0 255] in case of an 8-bit sensor.
3. Multi-resolution domain: the pre-processed image is transformed
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(c) (d)
(g) (h)
into the wavelet domain. In this work, the Dual-Tree Complex
Wavelet Transform (DT-CWT) [1, 9] is used, a redundant multi-
scale representation, characterised by the near shift-invariance
and improved directional properties over the conventional Dis-
crete Wavelet Transform (DWT).
4. Denoising/contrast enhancement: simultaneous denoising and
contrast enhancement is performed on the wavelet coefficients as
described in Section 2.1.
5. The denoised and contrast-enhanced wavelet coefficients are
transformed back to pixel domain via Inverse DT-CWT.
6. Post-processing: In order to avoid clipping of the output image
pixels that fall outside of the 8-bit intensity range, a percentile-
based contrast compression is applied1 by scaling the pixels
smaller than the 25th percentile and larger than the 75th per-
observer in mind, a Wiener filtering (within 7 × 7 neighbour-
hood) and gamma correction (γ = 0.75) is applied to the output
image.
7. HSV to RGB conversion: analogously to the pre-processing stage
of the system, a HSV to RGB transform is performed, by com-
bining the original H, S and the modified V components.
Moreover, if the multiple images are available the still-image pro-
cessing can be extended to sequential 3-D processing by taking two-
frames (current and the next) averages of the estimated γ̂. The sim-
ulations with image sequences have confirmed that 3-D processing
leads to better overall denoising and enhancement results.
1 Online: Contrast stretching, http://tinyurl.com/contrast-stretching
 Table 1. Quality measurements of the enhancement results
method/metrics σnout EBCM EME
CLAHE 2.96 94.0 14.6
Contourlet 3.47 82.6 22.4
LAMCO 4.74 82.2 28.6
proposed 2.09 109.7 30.9
3. RESULTS AND DISCUSSION
In order to produce the data used in this work, first HeLa cells were
grown in a glass-bottom petri dish and loaded with EGF-Alexa488-
10nm gold and Transferrin-Alexa594-5nm gold [10]. The cells were
then imaged on a Perkin Elmer spinning disc confocal microscope,
acquiring 1 frame per second at a 512 × 512 resolution.
The resulting images are compared both qualitatively and quan-
titatively to those obtained with other state-of-art methods, such as
CLAHE [2] and our implementations of curvelet-based CE [8] and
Locally Adaptive Multiscale Contrast Optimisation (LAMCO) [6].
We judge the results in terms of subjective perceptual quality and
objective quality measurements, such as output noise standard devi-
ation σnout , Edge-Based Contrast Measure (EBCM) [3] and Entropy
Measure of Enhancement (EME) [11].
Figure 2 shows the output of the tested methods and output at
particular stages of the proposed method. By comparing Figure 2
(b), (c) and (d) it can be observed that the combination of both de-
noising and CE is necessary for successful image restoration. Com-
parison of Figure 2(d) and (h) reveals that 3-D processing preserves
better the sharp details of the image due to consistency of some pat-
terns present in the two adjacent frames while suppressing incidental
noise. The remaining methods suffer above all from extensive ampli-
fication of the noisy background. These observations are confirmed
by quantitative measures as shown in Table 1. The proposed method
results in the lowest noise level (σnout ) and achieves the highest score
according to both edge- (EBCM) and entropy-based (EME) contrast
enhancement measures. Finally, Figure 3 shows a preliminary nu-
clei detection result as proposed in [12]. It can be clearly seen that
the image enhanced with our method generates much fewer spurious
detections while extracting the most prominent nuclei locations.
4. CONCLUSION
A novel method for preprocessing of microscopy images based on
the local statistics of their wavelet coefficients has been proposed in
this paper. The images are simultaneously contrast-enhanced and de-
noised by non-linearly re-scaling the wavelet coefficients modelled
as the bivariate Cauchy distribution. The enhancement methodology
proposed does not require user interaction and is shown to improve
significantly the readability of light microscope images. Apart from
improvement in terms of the perceptual quality over other state-of-
art contrast enhancement methods, the method is shown to facilitate
some biomedical imaging tasks such as nuclei detection.
In future work, it would be interesting to explore further the 3-
D information with the use of trivariate statistical models. The re-
sults of the proposed method should also be further assessed in con-
junction with other biomedical imaging applications, for example by
vesicle tracking in cellular bioimages.
5. REFERENCES
[1] Artur Loza, David R. Bull, Paul R. Hill, and Alin M. Achim,
“Automatic contrast enhancement of low-light images based
1086
(a) original image (b) denoised & enhanced image
Fig. 3. Example of nuclei detection
on local statistics of wavelet coefficients,” Digital Signal Pro-
cessing, vol. 23, no. 6, pp. 1856 – 1866, 2013.
[2] Etta D. Pisano et al., “Contrast limited adaptive histogram
equalization image processing to improve the detection of sim-
ulated spiculations in dense mammograms,” J. Digital Imag-
ing, vol. 11, no. 4, pp. 193–200, 1998.
[3] T. Celik and T. Tjahjadi, “Automatic image equalization
and contrast enhancement using Gaussian Mixture Modeling,”
IEEE Transactions on Image Processing, vol. 21, no. 1, pp.
145–156, Jan. 2012.
[4] Jian Lu and D.M. Healy, “Contrast enhancement via multi-
scale gradient transformation,” in Image Processing, 1994.
Proceedings. ICIP-94., IEEE International Conference, Nov.
1994, vol. 2, pp. 482–486 vol.2.
[5] Xuli Zong, A.F. Laine, and E.A. Geiser, “Speckle reduction
and contrast enhancement of echocardiograms via multiscale
nonlinear processing,” IEEE Transactions on Medical Imag-
ing, vol. 17, no. 4, pp. 532–540, Aug. 1998.
[6] N. Bonnier and E.P. Simoncelli, “Locally adaptive multiscale
contrast optimization,” in IEEE International Conf. on Image
Processing ICIP 2005, Sept. 2005, vol. 1, pp. I–949–52.
[7] A. Mencattini, F. Caselli, M. Salmeri, and R. Lojacono,
“Wavelet based adaptive algorithm for mammographic images
enhancement and denoising,” in ICIP-05 (IEEE Int. Conf. on
Image Processing), Sept. 2005, vol. 1, pp. I–1141–4.
[8] Kang Li, Xuejun Chen, Xiangjiang Hu, Xiang Shi, and Long
Zhang, “Image denoising and contrast enhancement based on
nonsubsampled contourlet transform,” in IEEE Int. Conf. on
Comp. Science and Inf. Technology, 2010, vol. 2, pp. 131–135.
[9] N G Kingsbury, “Image processing with complex wavelets,”
Phil. Trans. R. Soc. Lon., vol. 357, pp. 2543–2560, Sept. 1999.
[10] Edward Brown, Jan van Weering, Thom Sharp, Judith Man-
tell, and Paul Verkade, “Capturing endocytic segregation
events with HPF-CLEM,” Correlative Light and Electron Mi-
croscopy, vol. 111, pp. 175–201, 2012.
[11] S.S. Agaian, B. Silver, and K.A. Panetta, “Transform coef-
ficient histogram-based image enhancement algorithms using
contrast entropy,” Image Processing, IEEE Transactions on,
vol. 16, no. 3, pp. 741–758, 2007.
[12] Khuloud Jaqaman et al., “Robust single-particle tracking in
live-cell time-lapse sequences,” Nat. Meth., vol. 5, no. 8, pp.
1548–7091, 2008.