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Advanced Image Processing Techniques and Applications for Biological Objects
Adem Alpaslan Altun
Department of Computer Engineering
Faculty of Technology, Selcuk University
Selçuklu, Konya/TURKEY
e-mail:altun@selcuk.edu.tr
Abstract— In this article, modern technical means of obtaining,
processing and analysis of images for biological objects are
studied. Whole process from recording the image, to
processing it, is dissected both in hardware and technical
details. Information about automated systems of processing for
general purpose researched, and also confocal laser scanning
microscope specialized system of image processing and
techniques behind the analysis for micro objects are considered.
The confocal approach made it much easier to obtain images of
living microobjects, made it possible to automate the
accumulation of three-dimensional data, and improved images
of drugs using simultaneously several fluorescent markers.
Keywords-advanced image acquisition, processing techniques
and Applications, image processing, image analysis.
I. INTRODUCTION
The purpose of this study is Nondestructive testing (NDT)
provides safe operation to engineering components – it
eliminates the risk of damage during operation, and does not
require specific sample preparation. It has been widely used
to detect and evaluate defects, or measure properties of
different types of materials and engineering structures [1].
Examples of NDT techniques include ultrasonic, radiography,
infrared thermography, electromagnetic techniques and
visible optical methods. The imaging principles and imaging
facilities used by these techniques can be very different, but
almost all the techniques listed above require image
processing to some extent. In this chapter, instead of
extensively exploring all the NDT techniques and
corresponding image processing methods, we will focus on
optical measurement technique and related image processing
methods, and use porous materials as specimens [2].
Optical microscopy and digital camera imaging are two
examples of optical measurement technique, where optical
microscopy is the conventional one. Based on how the light
transfers from the sample to the objective, it can be
categorized to two different modes – transparent mode and
reflected mode. Digital camera imaging appeared with the
development of semiconductor industry. Digital images
bring convenience for image storage, image transferring, and
subsequent image processing. Actually, digital image
acquisition facilities, e.g. Complementary Metal Oxide
Semiconductors (CMOS) and Charge Coupled Device
(CCD), can be combined with the traditional microscope to
form digital microscopes [3]. As a convenient way of
measurement, optical microscope and digital camera imaging
978-1-5386-2030-4/17/$31.00 ©2017 IEEE
Anar Taghiyev
Graduate School of Natural and Applied Sciences
Selcuk University
Selçuklu, Konya/TURKEY
e-mail:gsimple07@hotmail.com
system has been extensively used in the area of
microelectronics, nanophysics, biotechnology,
pharmaceutical research, mineralogy, and material science[4].
Typical image processing procedures include image
acquisition, image alignment/stitching, image contrast
enhancement, grayscale thresholding, and/or image
subtraction. The characteristic of the specimens determines
which image processing techniques to be used. For instance,
for the images with background noise, image subtraction can
be applied when the background can be evaluated; otherwise
thresholding might be applied to eliminate the background
influence [5].
II. AUTOMATED IMAGE PROCESSING SYSTEM FOR
GENERAL PURPOSE
Automated image processing system (AIPS) is a complex
of hardware and software intended for recording, storing,
displaying processing and storage of digital images. The
individual components of the AIPS can be physically
separated in space and time, but each is fundamentally
necessary to complete the cycle of digital image processing
[6].
The first step in any digital image processing system is
the recording of images. This is a two-step process in which
a photosensor (or simultaneously a plurality of photosensors)
and a signal converter are involved in a digital form. There
are many different types of sensors, for instance, based on
point, line or matrix scanners. The purpose of the
photosensor is to generate an electrical signal at output,
which is a two-dimensional array of intensities of the
processed image. The most commonly used sensor is a video
camera [7].
The camcorder converts the optical image to an analog
signal. It uses a lens to focus light rays on a two-dimensional
photodetector. The photodetector converts light energy into a
proportional electrical signal. The electrical signal is the
analog form of the image signal [8].
After the camera is used an analog-to-digital converter
(ADC), which, as its name implies, converts an analog signal
to digital form. The ADC can be a separate device or an
integral component of a video camera or an image processor
itself. The ADC samples the video signal values uniformly
along each video line of the sweep, producing an array of
digital values that map the optical image to computer
memory, as shown in Fig. 1.


Figure 1. The use of radio waves for imaging by nuclear magnetic
resonance (NMR)
Each discrete (separate) value in the digital image
represents the average light intensity measured over the
sampling interval of the digital converter and is referred to as
an image element or pixel (that is "picture element"). Pixel is
the smallest integral part of a digital image. The properties of
the optical digital converter determine how accurately the
digital image corresponds to the original optical image (Fig.
2). Then the digital image is transferred for storage in digital
memory, which is a high-speed semiconductor device. In
digital memory, the image becomes physically accessible for
subsequent digital processing and analysis operations (Fig. 3).
A digital image contains a very large amount of computer
information, which usually needs to be processed all at once.
In a typical digital image containing 512 rows of 512 pixels
in each, 8 bits (1 byte) of computer memory is required for
one pixel. This corresponds to 262,144 bytes of computer
memory. Since it is often necessary to store several images
in memory at the same time so that they can be compared,
the required memory can be several times larger. To this end,
most imaging systems have two different digital memory
devices: digital image memory for storing one or more
processed digital images and the main computer's memory
used to store programs and other data [9].
When the main computer can not store all of the entire
digital image in memory, it moves small portions of the
Figure 2. Analog-to-digital image conversion

image back and forth between the storage device and its own
memory to execute the processing programs. With large
images or insufficient computer resources, this process can
be quite long. The delay caused by the limitations of the
main computer may be unacceptable when real-time image
processing is required.
Much faster processing can be implemented in special
purpose computers using special matrix or video processors
that can operate with very large numeric arrays. Such
processors can very quickly perform limited types of
arithmetic or logical operations with data in their memory.
Using a matrix processor to manipulate digital images, the
host computer only controls its operation and does not
control the image memory itself [10].
Typically, the matrix processor and the digital memory of
the images it manages, along with additional memory areas,
for storing conversion tables and other data used in the
processing of digital images are combined in one device.
Physical devices that are used to store digital images, but do
not operate with them, are called image buffers [11].
Once the processed image is received, it can be sent to
the display for visualization and to memory for storage. A
digital image can be converted to an analog video format
using a Digital-to-Analog Converter (DAC). It can be
displayed on a monitor with high resolution, sent to a photo
printer or stored on an optical or magnetic media. To ensure
maximum image reproduction quality, the video monitor
parameters must be equal to or better than the spatial and
photometric resolution of the optical digital converter of the
image recording system [12].
Control over the operation of the AIPS and all stages of
image processing is carried out with the help of the master
(main) computer. It also provides an interface with the user.
A controlling computer can process a digital image stored in
memory. Further, if necessary, the control computer can
transfer the image from its memory to other computers,
various network devices or to external mass storage devices
on magnetic or optical media. For small autonomous AIPS, a
specialized microcomputer or a personal computer is often
used as the main computer. More applications that are
complex require specialized workstations [13].

Figure 3. The main components of AIPS for general purpose
Despite the fact that modern universal computers have
enough computing resources to perform any conceivable
operation on the image stored in its memory, the speed of its
execution can be limited. With large images, this process can
be quite long. The delay caused by the limitations of the host
computer may be unacceptable when real-time image
processing is required (for instance, directly during the
medical examination or experiment). To increase the speed
of image processing, AIPS includes specialized high-speed
digital image processing processors optimized for operations
with two-dimensional data sets [14].
Universal AIPS - the concept is largely conditional. It is
inexpedient to produce AIPS for "all cases of life", therefore
the most widespread are specialized systems, such as
tomographs, ultrasound devices, etc. In practice, AIPS can
have a variety of architectures, using a combination of
hardware and software needed to solve specific applications.
The most important component of AIPS is software that
implements the technology of the system. Depending on the
specialization of AIPS, the set of available functional
program modules can vary greatly. As a rule, the software
includes the following most frequently used modules:
· Data input module (input of frames and series of frames,
registration of input data);
· Module for editing and transformation (editing,
changing brightness, contrast, morphological operations,
arithmetic operations, special filters, etc.);
· The recognition module (finding the analyzed objects
on the image);
· The module of measurements (measurements in
interactive and automatic mode, classification of objects,
statistical processing, etc.);
· The output of the results (output of the results of
processing, printing of the conclusion form, exchange with
the database, interaction with other programs, recording
images to disk, etc.);

· The module for configuring the program (setting
function keys, creating algorithms, setting the initial
parameters of the program, etc.);
· System of prompts and reference information.
In addition, the software may include specialized
modules for controlling the formation and input / output of
images.
Currently, many standard free and commercial software
packages for digital image processing have been developed.
These programs contain various digital image-processing
operations and are designed for use on general-purpose
computers [15].
Another important component of AIPS is its
methodological support, which implements the technology of
analysis of biomedical objects in a specific applied field:
methods for preparing standard preparations, forming,
processing and analyzing their images, interpreting and
recording results. Methodological support for a specific field
of research, as a rule, is a generalization of long-term studies
of a large number of highly qualified specialists [16].
The methodological support is included in the AIPS in
the form of manuals and predefined techniques - the
sequence of the image processing operations performed in
the automatic mode, which, in some cases, can be modified
or supplemented. The predefined techniques correspond to
the field of application of AIPS, for instance a set of
techniques for typing chromosomes.
III. SPECIALIZED SYSTEM OF IMAGE PROCESSING AND
ANALYSIS
The concept of confocal microscopy was proposed in the
mid-1950s. by Marvin Minsky. Today, laser scanning
confocal microscopy has become an important tool for
structural and functional studies of cells and tissues.
The term "confocal" means that a confocal diaphragm of
a small size is placed in the optical plane, conjugated with
the focal plane of the lens. The light emitted by the analyzed
point of the drug passes through the diaphragm and is
recorded, and the diaphragm mainly retains the light from the
remaining points. In this case, the illuminator does not create
a uniform illumination of the field of view, but focuses the
light at the analyzed point. This design allows you to record
the signal only from a thin layer of the drug. A schematic
diagram of the confocal microscope is shown in Fig. 4.
Confocal microscopy has several advantages over
traditional optical microscopy, including a shallow depth of
field, elimination of the focus-free "parasitic" illumination
that reduces contrast, and the ability to obtain serial optical
sections (slices) from thick-layered preparations. In
biomedical research, the main application of confocal
microscopy is in the production of images of fixed or living
cells and tissues that are usually labeled with one or more
fluorescent probes [17].
When an image of a fluorescent preparation is obtained
using a conventional optical microscope, the secondary
fluorescence emitted by the regions of the drug lying outside
the field of view is often mixed with the fluorescence from
the focusing region. This situation is especially characteristic
for preparations having a thickness greater than 2 μm. The
confocal method of image formation leads to a significant
improvement in resolution both in the plane of the drug and
in the axial direction, due to the possibility to exclude from
the image out-of-focus information that occurs in thick
fluorescently marked samples.
Modern models of confocal microscopes are relatively
easy to manage and have become part of the basic toolkit of
research centers for processing images of collective use. The
resolution achieved in a Laser Scanning Confocal
Microscope (LSCM) is somewhat better than in a
conventional light-field optical microscope, but still worse
than the transmission electron microscope resolution. This
allowed the LSCM to fill in some degree the free niche
between the two traditionally used microscopy methods. Fig.
4 shows the schematic diagram and Fig. 5 shows the
functional diagram of the optical system LSCM.
Figure 4. Optical scheme of a laser scanning confocal microscope
Figure 5. Functional diagram of laser scanning confocal microscope
In a traditional microscope, which can be called a wide-
field microscope, as opposed to a confocal one, a mercury or

xenon lamp illuminates the whole field of view, and the
image formed can be directly viewed by the eye or projected
onto a recording device or film. The imaging method in a
confocal microscope differs significantly. Illumination of the
drug is achieved by scanning with one or more focused laser
light beams (Fig. 6). Images of the preparation obtained in
this way are called optical sections or sections. This
terminology refers to the non-destructive method by which
the tool obtains serial slices using focused light, rather than
physical means of cutting the preparation [18].
The confocal approach made it much easier to obtain
images of living microobjects, made it possible to automate
the accumulation of three-dimensional data, and improved
images of drugs using simultaneously several fluorescent
markers (Fig. 7).
Figure 6. Restoration of the 3-dimensional structure of the preparation
Figure 7. An example of reconstructing a yeast cell image by the serial
optical reconstruction method (Janos Demeter and Shelley Sazer,
Department of Biochemistry, Baylor College of Medicine, Houston, Texas).
(A) the original image, (B) restored
The optical section is the basic unit of the image in
confocal microscopy. Images can be obtained from fixed and
colored preparations using one-, two-, three-, or multi-wave
illumination modes, using simultaneously several lasers to
excite fluorescence. Minor registration errors are corrected
by digital image processing techniques [19].
Most of LSCMs require approximately 1 second. To
register one optical section. To improve the signal-to-noise
ratio, averaging several frames of the same section is usually
used. The accumulation time of the image depends on the
image size in pixels and the performance of the computer
system.
Several images can be extracted from the z-series,
passing through the area of interest of the preparation, and
processed to isolate the micro objects under investigation.
Based on the Z-series of optical sections, the deconvolution
method can be used to reconstruct the three-dimensional
structure of the analyze, as shown in Fig. 6.
Deconvolution analysis is a technique that allows
processing of a stack of (Z-series) images of a micro object
obtained along the optical axis of a microscope. To do this,
the microscope must be equipped with a stepping motor
connected to the lens focusing system to ensure that images
are obtained at precisely defined intervals between the focal
planes in the preparation. In a typical application,
deconvolution analysis is used to eliminate scattered light
and remove out-of-focus light from a specific focal plane.
Deconvolution analysis can be applied to the entire stack of
images for constructing projections or a three-dimensional
model of a micro object [20].
To visualize the reconstructed micro object, you can
interactively set various parameters of the texture, color,
viewing angle of the display. The possibility of switching on
and off in the resulting image of various structures makes it
possible to separately examine the parts of the micro object.
A virtual space camera, freely movable around a 3-
dimensional model, allows the observer to look inside the
micro-object, studying its internal structure.
IV. CONCLUSION
One of the most important rules in the task of image
reconstruction is to avoid processing data containing any
discontinuities of continuity, of which cutoffs and
truncations are most undesirable, since in their presence
there are usually false details (often called artifacts,
especially in medical Applications). Thus, generally, it is
desirable to pre-process the image in order to completely
compensate for all the gaps and other removable defects that
are possible in them. However, if the gaps are not eliminated,
then the corresponding artifacts, as a rule, prevail over any
additional noise introduced by the preliminary processing.
The most of common problems for confocal microscopy,
due to its high resolution and contrast, is the study of the
structure of cells and their organelles, for example, the
cytoskeleton, EPR, lysosomes, mitochondria, nucleus,
chromosomes and even genes. Co-localization in the cell of
two or more substances is also investigated. Another
problem is the study of dynamic processes occurring in
living cells. For instance, the cellular transport of
biologically active compounds, changes in the concentration
and distribution of calcium ions.
ACKNOWLEDGMENT
This work was supported by Scientific Research Projects
Coordinating Office of Selcuk University.

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