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 Optical Imaging
for Biomedical and
Clinical Applications
 Optical Imaging
for Biomedical and
Clinical Applications

Edited by
Ahmad Fadzil Mohamad Hani and
Dileep Kumar
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Library of Congress Cataloging–in–Publication Data

Names: Kumar, Dileep, 1985- author. | Hani, Ahmad Fadzil Mohamad, author.
Title: Optical imaging for biomedical and clinical applications / Dileep Kumar &
Ahmad Fadzil Mohamad Hani
Description: Boca Raton : Taylor & Francis / CRC Press, 2018. | Includes
bibliographical references.
Identifiers: LCCN 2017028543| ISBN 9781498750370 (hardback : alk. paper) |
ISBN 9781315368351 (ebook)
Subjects: | MESH: Optical Imaging--methods | Image Enhancement | Skin
Diseases--diagnostic imaging | Eye Diseases--diagnostic imaging
Classification: LCC R857.O6 | NLM WN 195 | DDC 616.07/54--dc23
LC record available at https://lccn.loc.gov/2017028543

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 Dedication
This book was made possible with the unwavering support of
all authors of the book chapters; former students—Leena, Dr.
Hermawan, Dr. Lila, Dr. Hanung, Toufique, Aamir Shahzad;
colleagues—Dr. Naufal, Dr. Norashikin, Dr. Nidal, Dr. Aamir, Dr.
Ibrahima, Professor Fabrice; and collaborators—Dr. Norashikin,
Dr. Suraiya, Dr. Adawiyah, Dr. Felix Yap, Dr. Nor Fariza.
Their dedication, perseverance and undying search for answers
during the course of the research work and clinical studies have
led to this piece of work. This book is dedicated to them.
Contents
Preface..................................................................................................................ix
Acknowledgements........................................................................................ xiii
Editors.................................................................................................................xv
Contributors.................................................................................................... xvii

Chapter 1 Introduction to optical imaging................................................ 1

Dileep Kumar and Ahmad Fadzil Mohamad Hani
Chapter 2 Skin image analysis granulation tissue for healing
assessment of chronic ulcers................................................... 23
Ahmad Fadzil Mohamad Hani and Leena Arshad
Chapter 3 Skin image analysis for vitiligo assessment........................ 59
Ahmad Fadzil Mohamad Hani, Hermawan Nugroho,
Norashikin Shamsudin and Suraiya H. Hussein
Chapter 4 Modelling and analysis of skin pigmentation................... 109
Ahmad Fadzil Mohamad Hani, Hermawan Nugroho,
Norashikin Shamsudin and Suraiya H. Hussein
Chapter 5 Retinal image enhancement and analysis for
diabetic retinopathy assessment........................................... 225
Ahmad Fadzil Mohamad Hani, Hanung Nugroho,
Lila Iznita Izhar and Nor Fariza Ngah
Chapter 6 Noise reduction of retinal image for diabetic
retinopathy assessment.......................................................... 291
Ahmad Fadzil Mohamad Hani, Toufique Ahmed Soomro,
Ibrahima Faye, Nidal Kamel and Norashikin Yahya
Chapter 7 Hyperspectral image analysis for subcutaneous veins
localization................................................................................ 359
Aamir Shahzad, Mohamad Naufal Mohamad Saad,
Fabrice Meriaudeau and Aamir Saeed Malik

Index................................................................................................................. 409

vii
Preface
We are motivated to compile the findings of several research and pre-
clinical observational studies at Hospital Kuala Lumpur and Hospital
Selayang that investigated the use of optical imaging techniques in der-
matology for monitoring of skin pigmentation diseases such as vitiligo
and in ophthalmology to enhance colour fundus images system in diag-
nosing retina-related eye sicknesses, such as diabetic retinopathy.
Optical imaging is an effective medical imaging technique for in vitro
and in vivo applications. It is yet a vast field, from the understanding of
effectiveness of the technique to the analysis of the images. It involves bio-
medical optics, photon propagation in tissues, bioluminescence and fluo-
rescence as well as hardware components that are required such as light
sources, filters and detectors. The discussion also includes various optical
microscopic imaging techniques along with the whole animal body imag-
ing technique specially used to image small animals.
Investigating the optical characteristics of ulcer tissues based on their
histology and cellular composition to detect their corresponding content
in ulcer colour images is central in developing algorithm that is able to
identify granulation tissue regions on the exterior of ulcers to provide an
objective assessment of the healing condition of chronic ulcers. This is
very significant in detecting early stages of ulcer healing especially where
granulation tissue is spreading slowly over the ulcer surface and cannot
be detected visually. This work essentially utilises the optical imaging
technique to characterise haemoglobin pigments and determining its con-
tent within and below the visible surface of ulcers. Identified regions of
haemoglobin distribution can then be utilised as image markers to iden-
tify areas of granulation tissue indicating the ulcer healing progression
and reflects on the efficacy of the ulcer management and treatment.
The physician’s global assessment of skin pigmentary skin disorders
such as vitiligo, requires visual inspection but pigmentation changes due
to treatment and takes 3 to 6 months to discern visually by the derma-
tologist. Therapeutic responses of vitiligo treatments can be different
from patient to patient and are typically very slow and time consuming.
Segmentation of vitiligo lesion areas can be performed after the separation

ix
x Preface

process that produces skin images due to melanin and haemoglobin only.
The repigmentation progression due to treatment of the lesion areas is
measured objectively. Measurements generate equivalent PGA scores that
are useful to physicians in evaluating the efficacy of the treatment in a
shorter time period for example in 6 weeks.
In colour fundus images, the low contrast between the blood vessels
and the varying contrast of its surrounding background makes it visually
difficult to determine the retinal vasculature accurately. In addition, fun-
dus images are found to have both multiplicative and additive noise, and
can contain artefacts. This contrast problem can be overcome by using
fundus fluorescein angiography (FFA) that creates fundus images of high
contrast; however, because of its invasive nature, injecting contrast agent
is not a preferred method. RETICA, a non-invasive image improvement
scheme is developed and addresses the issue of low and varying contrast
image, through RETINEX for contrast normalisation and ICA for contrast
enhancement.
With TDCE-RETICA, the presence of noise in the fundus image is also
addressed. The novelty of this technique is that the noise level has been
effectively reduced by TDCE. RETICA with TDCE provides a mecha-
nism to reduce noise and resolve low and varied contrast in colour fun-
dus images and provides an efficient and non-invasive manner for retinal
fundus image analysis and interpretation. It is a practical non-invasive
alternative to the invasive fluorescein angiogram for retinal imaging and
further analysis and interpretation for diagnosis and monitoring of vision
threatening complications.
It is a difficult task to localise suitable veins in patients having certain
physiological characteristics such as dark skin tone, deep veins and the
presence of scars, tattoos or hair on the skin. To overcome the problem
of difficult venous access, several techniques can be used. As NIR imag-
ing is considered to be the most suitable among the techniques in terms
of usability, cost and efficiency, the optimisation of NIR illumination in
order to overcome the difficulty of veins localisation for different skin
tone subjects is presented. Hyperspectral venous image data acquired
from 252 subjects, arrived at an optimised range of illumination wave-
lengths. It was concluded that the wavelength range of 800–850 nm is the
optimum range for illumination in NIR imaging for all skin tone subjects.
We hope the book addresses problems from the medical sciences to
engineering principles transcending disciplines and professions.

Ahmad Fadzil Mohamad Hani, FASc, FIEM, PEng, PhD

Dileep Kumar, PhD
Centre for Intelligent Signal and Imaging Research (CISIR)
Universiti Teknologi PETRONAS
Bandar Seri Iskandar, Perak.
Preface xi

MATLAB® is a registered trademark of The MathWorks, Inc. For product

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Acknowledgements
We would like to acknowledge Universiti Teknologi PETRONAS for
the financial, laboratory and programming support at CISIR (Centre
for Intelligent Signal and Imaging Research). We also acknowledge the
various university internal funds and external grants received from
the Malaysian Ministry of Science, Technology and Innovation, and
the Ministry of Higher Education for the research work. We would
like to thank our collaborators; dermatologists from the Department of
Dermatology, Hospital Kuala Lumpur and ophthalmologists from the
Department of Ophthalmology, Hospital Selayang.

xiii
Editors
Professor Ahmad Fadzil Mohamad Hani is an expert in the area of
image processing and computer vision. He graduated with a BSc (1st Class
Honours) in electronic engineering in 1983, earned his MSc in telemat-
ics in 1984 and PhD in image processing in 1991 from the University of
Essex, UK. He has been actively involved in machine vision and medical
imaging research since the early 1990s. His research activities range from
fundamental signal and image processing to pattern recognition to devel-
oping vision and image analysis applications in the biomedical imaging
area such as in retinal vasculature imaging for grading severity of dia-
betic retinopathy and digital analysis leading to objective assessment for
treatment efficacy of ulcer wounds and psoriasis lesions, and bio-optics for
skin pigmentation analysis. His current research challenges are develop-
ing new analysis techniques for early osteoarthritis and drug addiction
using MRI (magnetic resonance imaging)/MRS (magnetic resonance spec-
troscopy), and neuroergonomics using fNIRS (functional near-infrared
spectroscopy). He has authored over 200 research articles in journals and
conference proceedings, granted several patents and won several awards
for his work. He is a senior professor and heads the Centre for Intelligent
Signal & Imaging Research (CISIR), a Ministry of Higher Education Higher
Institution Centre of Excellence at Universiti Teknologi PETRONAS.
Professor Fadzil is a Fellow of the Academy of Sciences, Malaysia and
a Fellow of Institution of Engineers Malaysia. He is a registered profes-
sional engineer with Board of Engineers, Malaysia and a senior member of
the Institution of Electrical & Electronic Engineers Inc. He is a member of
the Governing Board of the International Neuroinformatics Coordinating
Facility (INCF). In industry, he is a member of the Board of Directors of
ViTrox Corporation Bhd., an R&D and public-listed company that develops
and manufactures automated vision inspection systems. He is also a mem-
ber of the Board of Directors of Prince Court Medical Centre, Kuala Lumpur.

Dr. Dileep Kumar is an expert in the area of biomedical imaging, focus-

ing on the development of image processing/analysis methods that are
incorporated into decision support systems/tools. He graduated with a

xv
xvi Editors

Bachelor of Technology (BTech) in information technology from Uttar

Pradesh Technical University (UPTU), Lucknow, India, in 2006, earned
his master of technology (MTech) from Indian Institute of Information
Technology, Allahabad, India in 2008 and PhD in electrical engineering
from the Universiti Teknologi PETRONAS, Malaysia in 2014. His cur-
rent research interests include medical image analysis, image processing,
pattern recognition, medical imaging, medical physics, bioengineering
and biomedical systems. In particular, quantitative analysis of biomedi-
cal image/signal data, technological development for the acquisition of
biomedical images and development of new algorithm/techniques have
been a focusing point of his research. He is currently working as research
scientist/manager at the Centre for Intelligent Signal & Imaging Research
(CISIR), a Ministry of Higher Education Higher Institution Centre of
Excellence at Universiti Teknologi PETRONAS. Dr. Dileep has published
more than 25 scientific articles; obtained 2 IPR’s, filed 6 patents and one of
his patents was granted in 2015. He has received ‘Best Researcher Award’
(1 award/year) at Universiti Teknologi PETRONAS in 2015, ‘Need Based
Award’ by Osteoarthritis Research Society International (OARSI) in 2013,
‘Young Investigator Collaborative Research Award’ in the year 2015 by
OARSI (one of the six recipients from all over the world and the first from
SEA) and ‘Young Engineers Award’, given by the Institution of Engineers,
India in 2010 (one of the three recipients). He has been actively involved
in the peer-review process of several high-ranked journals, serving as an
associate editor for the SM Journal of Orthopedics, served in the Technical
Program Committee in various international conferences and occupies in
the executive committee board of professional organisations.
Contributors
Leena Arshad Lila Iznita Izhar
Centre for Intelligent Signal and Centre for Intelligent Signal and
Imaging Research Imaging Research
Department of Electrical and Department of Electrical and
Electronic Engineering Electronic Engineering
Universiti Teknologi PETRONAS Universiti Teknologi
Malaysia PETRONAS
Malaysia
Ibrahima Faye
Centre for Intelligent Signal and Nidal Kamel
Imaging Research Centre for Intelligent Signal and
Department of Fundamental Studies Imaging Research
Universiti Teknologi PETRONAS Department of Electrical and
Malaysia Electronic Engineering
Ahmad Fadzil Mohamad Hani Universiti Teknologi
Centre for Intelligent Signal and PETRONAS
Imaging Research Malaysia
Department of Electrical and
Electronic Engineering Dileep Kumar
Universiti Teknologi PETRONAS Centre for Intelligent Signal and
Malaysia Imaging Research
Department of Electrical and
and Electronic Engineering
SIRIM Tech Venture Sdn Bhd Universiti Teknologi
Selangor PETRONAS
Malaysia
Malaysia
and
Suraiya H. Hussein
Damansara Specialist Hospital Department of Psychology
Damansara Utama University of Otago
Petaling Jaya Dunedin
Malaysia New Zealand

xvii
xviii
Aamir Saeed Malik
Centre for Intelligent Signal and
Imaging Research
Department of Electrical and
Electronic Engineering
Universiti Teknologi PETRONAS
Malaysia
Fabrice Meriaudeau
Centre for Intelligent Signal and
Imaging Research
Department of Electrical and
Electronic Engineering
Universiti Teknologi PETRONAS
Malaysia
Nor Fariza Ngah
Department of Opthalmology
Selayang Hospital
Selangor
Malaysia
Hanung Nugroho
Department of Information
Technology and Electrical
Engineering
Gadjah Mada University
Yogyakarta
DIY
Indonesia
Hermawan Nugroho
Faculty of Engineering
Computing and Science
Swinburne University of
Technology Sarawak
Malaysia
Mohamad Naufal Mohamad Saad
Centre for Intelligent Signal and
Imaging Research
Department of Electrical and
Electronic Engineering
Universiti Teknologi PETRONAS
Malaysia
Contributors
Aamir Shahzad
Centre for Intelligent Signal and
Imaging Research
Department of Electrical and
Electronic Engineering
Universiti Teknologi
PETRONAS
Malaysia
and
Department of Electrical and
Electronic Engineering
COMSATS Institute of Information
Technology
Islamabad
Pakistan
Norashikin Shamsudin
Department of Medicine
Faculty of Medicine and Health
Science
Universiti Putra Malaysia
Serdang
Malaysia
Toufique Ahmed Soomro
Centre for Intelligent Signal and
Imaging Research
Department of Electrical and
Electronic Engineering
Universiti Teknologi
PETRONAS
Malaysia
Norashikin Yahya
Centre for Intelligent Signal and
Imaging Research
Department of Electrical and
Electronic Engineering
Universiti Teknologi PETRONAS
Malaysia
chapter one

Introduction to optical imaging

Dileep Kumar and Ahmad Fadzil Mohamad Hani

Contents
1.1 Introduction................................................................................................ 1
1.2 Biomedical optics....................................................................................... 4
1.2.1 Background on photon propagation........................................... 4
1.2.2 Fluorescence and bioluminescence phenomenon..................... 6
1.3 Optical imaging hardware....................................................................... 6
1.3.1 Light sources................................................................................... 7
1.3.1.1 Broadband lamps............................................................ 7
1.3.1.2 Light emitting diodes..................................................... 7
1.3.1.3 Gas or solid-state lasers.................................................. 8
1.3.2 Filters used in optical imaging.................................................... 8
1.3.3 Photon detectors............................................................................. 8
1.3.3.1 Single-channel photon detectors.................................. 9
1.3.3.2 Multichannel photon detectors..................................... 9
1.3.4 Optical imaging modalities........................................................ 10
1.4 Optical imaging techniques based on microscopy............................. 14
1.4.1 Fluorescence microscopy............................................................ 15
1.4.2 Confocal microscopy................................................................... 15
1.4.3 Multiphoton imaging.................................................................. 16
1.4.4 Stimulated emission depletion.................................................. 17
1.4.5 Other techniques.......................................................................... 18
1.5 Optical imaging techniques for whole body animal imaging.......... 19
1.6 Summary................................................................................................... 21
References........................................................................................................... 21

1.1 Introduction
Medical imaging has brought revolutionary changes to the medical diag-
nostic field. In medical radiology, various imaging modalities are being
used to study the biological view of different anatomical and molecular
structures of human and animals for diagnostics [1]. Various imaging

1
2 Optical imaging for biomedical and clinical applications

modalities, such as x-ray, ultrasound and computed tomography (CT) that

work based on radiography for structural tissue visualisation are widely
used for imaging internal tissue organs, whereas magnetic resonance
imaging (MRI), positron emission tomography (PET), single-photon emis-
sion computed tomography (SPECT) and optical imaging that work based
on radiology are used for structural and molecular change measurements.
The mechanisms in these modalities, for example, x-rays and CT involve
radiation of x-rays to see through soft tissues [2]. In the case of ultrasound,
being an anatomical modality, it involves sound waves to measure differ-
ent anatomical structures of internal organs [3]. MRI involves interaction
of RF (radio frequency) energy with underlying tissues to obtain structural
and physiological features [4]. PET and SPECT falls under nuclear imag-
ing and these modalities are being used to measure chemical changes in
tissue using radioactive tracers [5]. Optical imaging uses the visible light
spectrum of electromagnetic radiation and its interaction with internal
tissues to evaluate molecular changes [6]. Each of the above modality has
its own advantages and limitations. A comparison of different imaging
modalities is given in Table 1.1.
Although most imaging modalities are able to visualise internal tis-
sues not all modalities are able to measure molecular changes associ-
ated with internal organs in humans and animals. Molecular changes
in underlying internal tissues can be visualised using MRI, PET, SPECT
and optical imaging techniques. However, imaging modalities such as

Table 1.1 Comparison between different medical imaging techniques

Soft
tissue Penetration Nonionizing Data
Modality contrast Resolution level radiation acquisition Cost
X-ray imaging Poor Excellent Organ- No Fast Low
tissue
Ultrasound Good Good Organ- Yes Fast Low
(US) imaging tissue
X-ray CT Good Excellent Organ- No Slow Moderate
tissue
MRI and its Excellent Excellent Organ- Yes Slow High and
derivatives tissue very high
PET Excellent Low Tissue- No Slow Very high
cellular-
molecular
SPECT Excellent Low Tissue- No Slow Very high
cellular
Optical Excellent Moderate Tissue- Yes Fast Low
imaging cellular-
molecular
Chapter one: Introduction to optical imaging 3

PET and SPECT involve the use of contrast agent or radioactive trac-
ers that make these modalities invasive. Nuclear imaging modalities
are widely used for animal imaging in preclinical environment [7].
MRI has the potential to visualise internal tissues as well as measure
molecular changes corresponding to the internal tissues in the body.
However, MRI for the measurement of chemical changes requires con-
trast enhance agents that is injected prior to MR (magnetic resonance)
scanning in ­subjects [8]. So far, optical imaging modalities using visible,
ultraviolet (UV) and infrared light and the special properties of photons
are noninvasive and have proven to be effective in visualising details
of internal tissues and organs as small as cells and molecules level. It
also takes advantage of various colors of light to visualise and measure
several properties of tissue at the same time while other modalities are
unable to do so [6].
Optical imaging techniques can be broadly classified as biolumines-
cence imaging and fluorescence imaging [9]. Bioluminescence imaging is
used for imaging molecules in small animals where the light is emitted
into living organisms. The discovery of fluorescence and fluorescence
microscopy has been instrumental in imaging the single-cell structures
at the microscopic levels with the help of microscopic lenses. Fluorescent
images are obtained using a light source of specific wavelength to excite
the targeted molecule, which in turn emits light of longer wavelength
than absorbed. This emitted light is used to generate fluorescent optical
images. Optical imaging techniques being investigated meet the chal-
lenges and improvement in molecular imaging in preclinical examina-
tion and patient concern. Combination of targeted molecules in vivo and
optical contrast agents imaging sensitivity are driven in parts for molec-
ular imaging in order to emphasise an optical imaging systems [10]. By
combining optical imaging techniques such as bioluminescence imaging
and fluorescence with near-infrared (NIR) spectrum, the signal-to-back-
ground ratio for detecting specific molecular signals can be increased and
similarly can be achieved with other molecular imaging modalities. In
advanced cases, a fundamentally simplified gene expression imaging is
produced using bioluminescent and fluorescent proteins that act as syn-
thesised optical active biomarkers. It is also noted that optical imaging
with its advancements can now be used in clinical practices for some
applications and it is widely investigated for future research directions
toward its application in clinics.
In this chapter, biomedical optics, photon propagation in tissues, bio-
luminescence and fluorescence are discussed followed by the parameters
and components required for optical imaging such as light sources, filters,
and detectors. In addition, microscopic imaging techniques for whole ani-
mal body imaging using optical imaging are discussed.
4 Optical imaging for biomedical and clinical applications

1.2 Biomedical optics

1.2.1 Background on photon propagation
In optical imaging, electromagnetic radiation ranging from 400 to 700 nm,
referred to as the visible light spectrum, is used to produce images through
microscopy, endoscopy and colonoscopy for biomedical applications [11].
The range of spectrum can be extended to the soft UV (short wavelengths)
and NIR (long wavelengths) ranges in order to perform advanced optical
imaging such as fluorescence and multispectral imaging. Unlike x-rays
with wavelengths ranging from 0.01 to 10 nm, which are able to pene-
trate deep through tissues, visible light spectrum used in optical imag-
ing interacts with tissues to a certain depth. Light interaction is defined
in terms of reflection, refraction, diffusion, interference propagations, etc.
Parameterisation of optical photon propagation is in terms of total emis-
sion or reflection within a solid angle.
Whenever a photon beam is incident on biological tissue, scattering
or absorption phenomenon is observed and can be measured in terms of
scattering coefficient, µs and absorption coefficient, µa. This is referred to
as the transport coefficient (also termed as extinction coefficient or total
interaction coefficient) and is given by

µt = µs + µa (1.1)

For biological tissues (also considered as turbid medium), the absorp-

tion coefficient is insignificant compared to scattering coefficient, that is,
µa  µs; thus, the transport coefficient, µt = µs. If the photons undergo
several scattering events, then the scattering coefficient is reduced as
expressed by

µs′ = µs (1 − g ) (1.2)

Here, g is the scattering anisotropic coefficient, which is typically in

the range of 0.8–1.
Absorption coefficient is defined as the probability of photon absorp-
tion in a medium per unit path length. The reciprocal of absorption coef-
ficient is called the mean absorption length. Optical parameters for the
biological tissues are wavelength-dependent. For example, different lay-
ers of skin have different absorption coefficients with respect to the wave-
length of optical photon beam, as shown in Figure 1.1.
The scattering coefficient, µs, is defined as the probability of photon
scattering in a medium per unit path length. Biological tissues typically
have a scattering coefficient of 100 cm−1. The inverse of µs is the scatter-
ing mean free path (MFP). Biological structure interaction with light leads
Chapter one: Introduction to optical imaging 5

Optical properties of seven layers in skin

102
Stratum corneum
Epi
Papillary
Upper
Absorption coefficient (log10 (ua))

Reticular
Deep
101
Fat

100

10–1
400 500 600 700 800 900 1000
Wavelength (nm)

Figure 1.1 Wavelength vs. absorption coefficient for seven layers of skin.
(Adapted from A. P. Dhawan, B. D’Alessandro, and X. Fu, Biomedical Engineering,
IEEE Reviews in, vol. 3, pp. 69–92, 2010.)

to optical scattering ranging between whole cells and membrane [11].

Scattering of light is inversely proportional to the wavelengths but more
precisely it remains comparatively stable in the visible range spectrum.
The MFP is given in terms of scattering and absorption coefficients as
reciprocal of transport coefficient as expressed by

1
MFP = (1.3)
µt

With, µt = µs + µa and µa  µs,

1
MFP = (1.4)
µs

Transport TMFP in terms of µs′ for which the beam has undergone
several scattering is expressed by

1
TMFP = (1.5)
µs′
6 Optical imaging for biomedical and clinical applications

This generally holds for most tissues assuming that scattering is

dominant over absorption, that is, µa  µs′. Therefore, the relationship
between two parameters is given as MFP = TMFP (1 − g); higher values
of g increases forward scattering of light with longer duration to diffuse,
leading to in-depth penetration using microscopic technique [12].

1.2.2 Fluorescence and bioluminescence phenomenon

Fluorescence is a phenomenon in which a molecule emits light when
it returns to the ground state after being excited by an external light
source [13]. Light produced by the molecule while relaxing to the ground
state is of a longer wavelength relative to the wavelength of the excita-
tion light. The duration of time for which the molecule is in excitation
state is considered as the lifetime of the molecule. The material’s ability
of absorbing light photons of a particular wavelength and entering into
excitation state and emitting the light of longer wavelength while return-
ing to ground state is called the fluorescence property of the material.
The fluorescence process can be divided into three steps: (1) excitation of
molecule to new state by an external light source of specific wavelength
within certain femtoseconds, (2) vibrational relaxation state in the excited
mode for certain picoseconds and (3) molecule emitting light photons at
a longer wavelength while returning to the ground state, which requires
some nanoseconds [13]. The entire molecular fluorescence lifetime from
excitation to relaxing to ground state is measured in billionths of a second.
Such phenomenon is used by many of the fluorescent microscopic imag-
ing devices for optical imaging of the biological tissues.
Bioluminescence is the phenomenon of bio-organisms producing light
by the enzymatic reaction of a luciferase enzyme with its substrate lucif-
erin. This luciferase enzyme is obtained from a firefly, which is a natu-
ral source of luciferase and is widely used in bioluminescence process
to measure molecular changes in tissues of small animals. Luciferin is
injected into a small animal and passes through blood tissues including
the brain and placenta causing emissions of light from the molecules of
small animals. The emitted light reaches its peak after 10–12 min and loses
its intensity slowly over 60 min [10]. This time frame taken to lose emitted
light intensity is sufficient to capture microscopic images of these molecu-
lar tissues in small animals. There are many natural sources of luciferase
enzyme from other organisms that produce light of different wavelengths
possibly leading to different colors such as red, green, blue, etc. [10].

1.3 Optical imaging hardware

An optical imaging system consists of three main components: (1) light
source that is being emitted into tissues and reflected in the form of
Chapter one: Introduction to optical imaging 7

images, (2) filters form an integral part of optical imaging system that gen-
erally removes any artefacts produced during acquisition, and (3) optical
detectors or photon detectors as the source component required to detect
the optical beams that is being reflected from light source after filtration.
The following subsections describe the three components of an optical
imaging system.

1.3.1 Light sources

Light sources for an optical imaging technique can be of any form but the
choice of a light source depends on the type of application. For biological
applications, the light source for optical imaging should be predictable,
stable, measurable and reliable [14]. It should also be adjustable to appro-
priate wavelengths, beam size and intensity for specific applications.
The most commonly used light sources for optical imaging in biomedi-
cal applications are lasers (gas and solid state), broadband lamps that are
capable of providing UV to NIR range of wavelengths and light-emitting
diodes (LEDs) [15].

1.3.1.1 Broadband lamps

Most commonly available broadband light sources are high-pressure arc
lamps and incandescent lamps. UV to NIR (200–1000 nm) intense broad-
band emission can be obtained from high-pressure arc lamps. These lamps
typically use xenon, mercury or mercury–xenon gases. These lamps are
to be handled with utmost care because of their explosive nature. Xenon
lamp has strong UV output while incandescent lamps cover the visible
range to NIR range. Xenon and incandescent lamps are the simplest light
sources and commonly used in quartz tungsten halogen lamp. The lat-
est available broadband light sources are supercontinuum light sources,
which generate light through propagation of high-power pulse via a non-
linear media. Most supercontinuum light sources can generate huge watt-
ages of optical power in a broad wavelength range from visible to NIR [15].

1.3.1.2 Light emitting diodes

LEDs are solid-state light sources based on semiconductors of p- and
n-type in a p–n junction, which emit light when both the terminals are
kept under different voltages [16]. Depending on the band gap of the p–n
junction, LEDs can cover full range of wavelengths from UV to NIR. LEDs
can generate different colours of light for various applications. LEDs offer
better SNR (signal-to-noise ratio) for vascular imaging applications since
haemoglobin has higher absorption in the visible range of wavelengths
below 650 nm [17]. Quantitative analysis of biomedical imaging involves
repeatable measurements and requires consistency; here, LEDs’ control-
lable intensity stability plays a vital role in such applications [18].
8 Optical imaging for biomedical and clinical applications

1.3.1.3 Gas or solid-state lasers

Lasers are light sources that can emit coherent and polarised light, which
exhibit monochromatic (peaked) spectrum. Optical imaging techniques
such as fluorescence and Raman spectroscopy most often use lasers.
Lasers can couple effectively with the optical fibres because of their sharp
and intense monochromatic type. Most commonly available lasers are gas
or solid-state lasers obtained using He–Ne (helium–neon), CO2 (carbon
dioxide), Ar+ (argon), nitrogen sapphire, Nd:YLF (neodymium–yttrium
lithium fluoride) and Nd: YVO4 (neodymium–yttrium orthovanadate)
solid states. The choice of lasers is made depending on the requirement of
wavelength, size, power and cost [15]. Lasers are the prime source of light
in the imaging techniques such as fluorescence imaging, optical tomog-
raphy, etc. Lasers are used to excite the fluorescence signals in biological
tissues in order to observe the abnormalities in the tissue through fluores-
cence imaging [13].

1.3.2 Filters used in optical imaging

Optical filters are the most common requirement of an optical imaging
system. Filters that are often used in optical imaging are notch filters (NF),
neutral density (ND), band-pass (BP), short-pass (SP) and long-pass (LP)
filters [19]. ND filters provide full band attenuation in applications with
very strong intensity for detectors. SP filter allows signals with smaller
wavelengths and attenuates longer wavelength light while LP filter allows
only longer wavelengths and attenuate shorter wavelengths. BP filters
allow the particular band (range) of wavelengths to pass and blocks the
rest but NF does the opposite. These filters can be narrowband or broad-
band depending on the size of the pass band.
Filters are mostly dependent on dispersion, interference or absorption.
Absorbing filters work on the principle of either transmitting or absorbing
the light. Interference filters work on the principle of either transmitting
or reflecting the light; none of the light is absorbed. Dispersive filters work
on the principle where the light incident on it is dispersed into different
wavelengths according to the application [19]. Optical filters are critical in
order to obtain sharp and high contrast images by blocking the scattered
light from reaching the detector. The scattered light received from the
unwanted tissues is blocked by optical filters in order to image the specific
tissue [20]. In biomedical optical imaging, optical filters are chosen based
on the requirements of the application and the type of light source used.

1.3.3 Photon detectors

Photon detectors are light sensors that sense light incident on it. There are
several types of detectors available for biomedical optical imaging and
Chapter one: Introduction to optical imaging 9

are generally classified into two groups namely, single- and multichannel
detectors. Depending on the application, the choice of detector is made by
considering several factors such as wavelength range, signal level, physi-
cal size and the data acquisition speed.

1.3.3.1 Single-channel photon detectors

The most commonly available single-channel photon detectors are PIN
diode, avalanche photo diode (APD) and photomultiplier tube (PMT)
[15]. PIN diodes are made up of silicon or In GaAs (indium gallium arse-
nide). Silicon photodiode operates in the range of 200–1100 nm and has a
peak response at 900 nm, whereas In GaAs operates in the range of 800–
1700 nm and has a peak response at 1500 nm. Signal is linearly depen-
dent on the input light and there is no requirement of any high voltage.
Because of very low sensitivity and small photoactive area, PIN photodi-
odes are often used in monitoring the excitation of intensity in fluores-
cence spectroscopy. However, the APD has a higher sensitivity compared
to PIN photodiode due to the avalanche multiplication that results in the
first stage of gain. A suitable APD is selected based on the range of wave-
lengths, size of detection area and electrical bandwidth [15]. In applica-
tions like fluorescence microscopy with very low signal detection level,
APDs are used because of its higher sensitivity and faster response time.
The PMT is made up of electron collector (anode), photon electron con-
verter (cathode) and electron multiplier. With higher bandwidth and gain,
PMTs are capable of photon counting in very low or short pulses of light.
PMTs are selected based on wavelength, beam size and intensity require-
ments. Most common photodetectors in PET scanners are PMTs, which
are used for small animal imaging [21].

1.3.3.2 Multichannel photon detectors

Multichannel detectors are preferred over single-channel detectors
because of its high quantum yield, multichannel capability and low dark
signal. Silicon-based charge coupled device (CCD) is the most preferred
detectors among multichannel photon detectors. There are three differ-
ent types of CCDs that are commonly used for optical imaging namely,
CCD with back illumination, CCD with front illumination and back-­
illuminated CCD with deep depletion, as depicted in Figure 1.2. The wave-
length operability range of these types are as follows: (1) front-illuminated
operates well in the range 400–1000 nm with maximum quantum peaked
at 700 nm, (2) back-illuminated operates in the range 250–1000 nm with
maximum quantum peak at 500 nm, (3) back-illuminated deep deple-
tion operates in the range 300–1000 nm with maximum quantum peak at
800 nm [15].
CCD with front illumination is useful for strong light detection while
CCD with back illumination is useful for the low-intensity light signals.
10 Optical imaging for biomedical and clinical applications

Silicon Silicon
dioxide Silicon dioxide

Incident Silicon Incident

light light

Polysilicon Thinned Polysilicon

gate silicon gate

Silicon
Silicon dioxide

Incident
light

Thinned Polysilicon
silicon gate

Figure 1.2 CCD sensor array types. (a) CCD with front illumination, (b) CCD
with back illumination and (c) back illuminated with deep depletion CCD.

Back-illuminated CCD with deep depletion is used for very low-intensity

light signals. In order to increase the speed of the sensors, the CCD multi-
sensors are combined with the light amplifiers often called as intensified
CCD (ICCD). The amplifier works as a shutter to intensify the light for the
speed gating. Thus, ICCDs are very fast operating detectors.

1.3.4 Optical imaging modalities

At present, various imaging modalities are available that use principles of
biomedical optics. The most commonly used biomedical optical imaging
techniques are optical coherence tomography (OCT), endoscopy, multi-
spectral endoscopy photoacoustic imaging, diffuse optical tomography
(DOT), diffuse reflectance imaging and microscopic techniques such as
fluorescence microscopy, stimulated emission depletion (STED), confocal
microscopy and multiphoton microscopy. These modalities are portable,
cost-effective, and are being used for in vivo and noninvasive imaging of
tissues, spanning from molecular and cellular levels. Example images of
these optical imaging techniques are shown in Figure 1.3a–d. A brief dis-
cussion of each modality is given below and in the following section. The
detailed discussion on microscopic optical imaging modalities is given in
Section 1.4.
Another random document with
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States, 'while specially asserting the rights of Hungary and
its independence.' Another version of this somewhat oracular
statement runs as follows:—'Hungary, without infringing the
Ausgleich law, will find ways and means of regulating those
affairs which, in virtue of the Pragmatic sanction, are common
to both States, while at the same time protecting her own
interests and giving greater emphasis to her independence.'
Dr. Szell added:—'When the right time comes I shall explain my
views, and eventually submit proposals to the House.
Meanwhile, let us husband our strength and keep our powder
dry.' The self-confident and almost defiant tone of this
forecast, coming from a responsible statesman accustomed to
display such prudence and moderation of language as M. Szell,
has made a profound impression in Austria. It assumes the
breakdown of the Austrian Parliamentary system to be a
certainty, and anticipates the adoption by Hungary of
one-sided measures which, according to M. Szell, will afford
more effective protection to its interests and confirm its
independence. This seems to be interpreted in Vienna as an
indication that the Hungarian Premier has a cut and dry scheme
ready for the revision of the Ausgleich in a direction which
bodes ill for Austria. The gravity of the Ministerial
statement is recognized by journals of such divergent views as
the semi-official 'Fremdenblatt,' the pan-Germanic and
Anti-Semitic 'Deutsche Zeitung,' and the 'Neues Wiener
Tagblatt,' which is the organ of the moderate German element.
The 'Neues Wiener Tagblatt' frankly acknowledges that, in
addition to all her other cares, Austria has now to consider
the crucial question of the form which her relations with
Hungary will assume at no distant date. Commercial severance
and declarations of independence are, it says, being discussed
by the initiated sections of the community in both countries,
as if it were a matter of merely economic concern, instead of
the greatest and most perilous political problem that the
Monarchy has been called upon to solve since the establishment
of the Dual system, which, in spite of its complexity, has
worked well for such a long period. The 'Neues Wiener
 Tagblatt,' nevertheless, admits that things have now reached a
stage at which economic severance is no longer impossible." In
a subsequent speech on New Year's Day, M. Szell declared that
it "would be a fatal mistake to sever the ties which had so
long connected the two countries, as the objects for which
they were called into existence still remained and their
fundamental basis was not shaken."

The Vienna journals, on that New Year's Day of 1901, reviewed

the past and surveyed the prospects of the future in gloomy
and pessimistic tones. Heading its article "Progress
Backward," the "Wiener Allgemeine Zeitung" said: "It is true
that Austria has at her disposal a larger and more efficiently
trained army than ever. The natural resources of the country
have been better developed than in the past. The progress of
the century has not been without influence upon ourselves.
But, whereas other nations are more vigorous, greater, and
mightier, we have become weaker, smaller, and less important.
The history of the world during the second half of the past
century has been made at our expense. … In the new partition
of the world no room has been reserved for Austria. The most
important events which will perhaps give the world a new
physiognomy are taking place without Austria's being able to
exercise the slightest influence thereon. We are living upon
our old reputation, but in the long run that capital will
prove insufficient."

AUSTRIA-HUNGARY: A. D. 1900 (December).

Census of Vienna.

See (in this volume)

VIENNA: A. D. 1900.

AUSTRIA-HUNGARY: A. D. 1901.
Parliamentary elections.
Weakening of the Clerical and Anti-Semitic parties.
Gains for the ultra-radical German parties.
Disorderly opening of the Reichsrath.
Speech of the Emperor from the throne.

From the parliamentary elections held in January the Clerical

and Anti-Semitic parties came back to the Reichsrath shorn of
about one-third of their strength, while the various radical
factions, especially those among the Germans, appear to have
made considerable gains. Even in the Tyrol, one of the
strongest of the Clerical leaders, Baron Di Pauli, was
defeated, and in Vienna the Anti-Semitic majority was cut to
less than one-fourth of what it had been three years before.
"The Pan-Germanic group," writes "The Times" correspondent
from Vienna, "which only numbered five in the last Parliament,
now musters 21. It will be remembered that it openly advocates
incorporation with the German Empire, and as a preparatory
measure the wholesale conversion of the German population of
Austria to Protestantism. It has hitherto been to a certain
extent boycotted by the other German parties, being excluded
from their so-called union for mutual defence and joint
action." "But the programme which had thus been boycotted by
the bulk of the German members has been the most successful of
all in the recent general election. The position of its
leading representative, Herr Schönerer, has been so
strengthened that he has been able to impose upon the whole
group the title of Pan-Germanic Union, and to enforce the
acceptance of the principle of 'emancipation from Rome.' The
latter demand caused a certain hesitation on the part of some
of his new followers, who, however, ultimately decided to
adopt it, although not to the full extent of renouncing the
Roman Catholic faith, as Herr Schönerer and his principal
lieutenant, Herr Wolf, themselves had done. At a conference of
the party its programme was declared to be the promotion of
such a federal connexion of the German provinces of Austria
with the German Empire as would furnish a permanent guarantee
for the maintenance of the German nationality in this country.
The party would oppose every Government that resisted the
realization of that object, and it could not participate in
 any manifestations of loyalty while such a Government policy
was maintained. At the same time, the party regarded it as
their obvious duty to emancipate themselves from Rome in a
political but not religious sense—that is to say, to free
themselves from the influence of the Roman Curia in affairs of
State.

{46}

"This boycotted party and programme now threatens to win the

voluntary or enforced adherence of the advanced section of the
other German groups which had hitherto declined to commit
themselves to such an extreme policy. The most moderate of all
the German parties, that of the constitutional landed
proprietors, has felt called upon to enter an energetic and
indignant protest against the foregoing Pan-Germanic
programme. While they are convinced supporters of the
Austro-German alliance, they unconditionally reject
aspirations which they hold to be totally inconsistent with
the tried and reliable basis of that agreement, and which
would constitute an undignified sacrifice of the independence
of the Monarchy. They further decline to make their
manifestations of loyalty to the Sovereign dependent upon any
condition; and they strongly condemn the emancipation from
Rome movement as a culpable confusion of the spheres of
religion and politics, and an infringement of the liberty of
conscience which is calculated to sow dissension among the
German nationality in Austria.

"It now remains to be seen to which side the bulk of the

German representatives will rally; to that of the Moderates,
who have re-affirmed their devotion to the Dynasty and the
existing Constitution, or to that of the Pan-Germanic
revolutionaries, who have decided to make their manifestations
of loyalty dependent upon the adoption by the Crown of their
programme.
"The outlook has thus undergone, if anything, a change for the
worse since the last Reichsrath was dissolved. The only
reassuring feature of the situation is that the fall of the
Ministry is not a primary end with any of the parties in the
Reichsrath. Dr. von Körber, who is a politician of great tact
and experience, has avoided friction on all sides."

The opening session of the newly elected Reichsrath was held

on the 31st of January, and the disorderly temper in it was
manifested upon a reference by the President to the death of
Queen Victoria, which called out cries of hostility to England
from both Germans and Czechs.

"In the course of the proceedings some of the members of the

Extreme Czech fraction warned the Prime Minister in
threatening terms against introducing a single word hostile to
the Czech nation in the coming Speech from the Throne. They
also announced their intention of squaring accounts with him
so soon as the Speech from the Throne should be delivered. The
whole sitting did not last an hour, but … what happened
suffices to show that not only the Pan-Germanic Union, but
also the Extreme section of the German People's party and a
couple of Radical Czechs, are ready at a moment's notice to
transform the Reichsrath into a bear garden."

On the 4th of February the two Houses of the Reichsrath were

assembled at the Palace and addressed by the Emperor, in a
speech from the throne of which the following is a partial
report: "His Majesty referred to various features of
legislation, including the Budget, the revision of the Customs
tariff, the promotion of trade, industry, and navigation, the
protection of the working classes and the regulation of the
hours of labour, the Government railway projects and the
Bosnian lines, and Bills for the regulation of emigration, the
construction of dwellings for the lower classes, the
repression of drunkenness, the development of the University
system and other educational reforms, and a revision of the
Press laws—in fact a whole inventory of the important
legislative arrears consequent upon the breakdown of
Parliament.

"The following passage occurs in the further course of the

speech: 'The Constitution which I bestowed upon my dominions
in the exercise of my free will ought to be an adequate
guarantee for the development of my people. The finances of
the State have been put in order in exemplary fashion and its
credit has been raised to a high level. The freedom of the
subject reposes upon a firm foundation, and thanks to the
scholastic organization and the extraordinary increase of
educational establishments general culture has reached a
gratifying standard, which has more especially contributed to
the efficiency and intelligence of my army. The Provincial
Diets have been able to do much within the limits of their
jurisdiction. The beneficial influence of the constitutional
system has penetrated as far as the communal administrations.
I am thus justified in saying that the fundamental laws of the
State are a precious possession of my loyal people.
Notwithstanding the autonomy enjoyed by certain kingdoms and
provinces, they constitute for foreigners the symbol of the
strength and unity of the State. I was, therefore, all the
more grieved that the last sessions of the Legislature should
have had no result, even if I am prepared to acknowledge that
such business as affected the position of the Monarchy was
satisfactorily transacted by all parties.'

"The Emperor then expressed his regret that other matters of

equal importance affecting the interests of Austria had not
been disposed of. His Majesty made an appeal to the
representatives of the Reichsrath to devote their efforts to
the necessary and urgent work awaiting them, and assured them
that they might count upon the Government. All attempts at the
moral and material development of the Empire were, he said,
stultified by the nationality strife. Experience had shown
that the efforts of the Government to bring about a settlement
 of the principal questions involved therein had led to no
result and that it was preferable to deal with the matter in
the Legislature. The Government regarded a generally
satisfactory solution of the pending language question as
being both an act of justice and a necessity of State.
Trusting in the good will manifested by all parties, the
Ministry would do its utmost to promote a settlement which
would relieve the country of its greatest evil. At the same
time, the Cabinet was under the obligation of maintaining
intact the unity of language in certain departments of the
Administration, in which it constituted an old and well-tested
institution. Success must never again be sought through
paralysing the popular representation. The hindrance of
Parliamentary work could only postpone or render quite
impossible the realization of such aspirations as most deeply
affected the public mind. The Sovereign then referred to the
damage done to the interests of the Empire by the obstacles
placed in the way of the regular working of the Constitution,
and pointed to the indispensable necessity of the vigorous
co-operation of Parliament in the approaching settlement of
the commercial relations between the two halves of the
Monarchy. The speech concluded with a warmly-worded appeal to
the representatives to establish a peace which would
correspond to the requirements of the time and to defend as
their fathers had defended 'this venerable State which accords
equal protection to all its peoples.'"

{47}

AUSTRIA-HUNGARY: A. D. 1901 (March).

Continued turbulence of the factions
in the Austrian Reichsrath.
Outspoken aim of the Pan-Germans.

At this writing (late in March), the disgraceful and

destructive conflict of reckless factions is still raging in
the Austrian Reichsrath, and the parties have come to blows
 several times. The hope of the German extremists for a
dissolution of the Empire seems to be more and more openly
avowed. On one occasion, "a Czech member, Dr. Sieleny, having
accused the Pan-Germans of wistfully glancing across the
frontier, Herr Stein, a member of the Pan-Germanic group,
replied, 'We do not glance, we gaze.' Being reproached with
looking towards Germany with an ulterior motive, the same
gentleman answered, 'You Czechs want to go to Russia, and we
Germans want to go to Germany.' Again, on being told that he
would like to become a Prussian, he exclaimed, 'I declare
openly that we want to go to the German Empire.' Finally, in
reply to another remark, Herr Stein observed that everybody in
the country who was an Austrian patriot was stupid."

----------AUSTRIA-HUNGARY: End--------

AUTONOMY, Constitutional:
Granted by Spain to Cuba and Porto Rico.

See (in this volume)

CUBA: A. D. 1897 (NOVEMBER);
and 1897-1898 (NOVEMBER-FEBRUARY).

AYUNTAMIENTOS.

Town councillors in Spain and in the Spanish American states.

See (in this volume)

CUBA: A. D. 1901 (JANUARY).

B.

BABYLON: Exploration of the ruins of the city.

See (in this volume)

ARCHÆOLOGICAL RESEARCH:
BABYLONIA: GERMAN EXPLORATION.
BABYLON: Railway to the ruins.

See (in this volume)

TURKEY: A. D. 1899 (NOVEMBER).

BABYLONIA: Archæological Exploration in.

See (in this volume)

ARCHÆOLOGICAL RESEARCH: BABYLONIA: AMERICAN
EXPLORATION.

BACHI,
BASHEE ISLANDS, The American acquisition of.

See (in this volume)

UNITED STATES OF AMERICA: A. D. 1898 (JULY-DECEMBER).

BACTERIAL SCIENCE, Recent.

See (in this volume)

SCIENCE, RECENT: MEDICAL AND SURGICAL.

BADENI, Count: Austrian ministry.

See (in this volume)

AUSTRIA-HUNGARY: A. D. 1895-1896.

BADEN-POWELL, General R. S. S.: Defense of Mafeking.

See (in this volume)

SOUTH AFRICA (THE FIELD OF WAR):
A. D. 1899 (OCTOBER-NOVEMBER); and 1900 (MARCH-MAY).

BAGDAD, Railways to.

See (in this volume)

 TURKEY: A. D. 1899 (NOVEMBER); and JEWS: A. D. 1899.

BAJAUR.

See (in this volume)

INDIA: A.D. 1895 (MARCH-SEPTEMBER).

BALFOUR, Arthur J.:

First Lord of the Treasury in the British Cabinet.

See (in this volume)

ENGLAND: A. D. 1894-1895; and 1900 (NOVEMBER-DECEMBER).

BALFOUR, Arthur J.:

Tribute to Queen Victoria.

See (in this volume)

ENGLAND: A. D. 1901 (JANUARY).

BALKAN AND DANUBIAN STATES, The.

"The States of the Balkan Peninsula, ever since the practical

disruption of European Turkey after the war of 1877-78, have
been in a condition of chronic restlessness. Those who desire
the repose of Europe have hoped against hope that the new
communities which were founded or extended on the ruins of the
Ottoman dominion in Europe would be able and willing to keep
the peace among themselves and to combine in resisting the
intrusion of foreign influences. These expectations have been
too frequently disappointed. The lawlessness of Bulgaria and
the unsettled state of Servia, more especially, continue to
constitute a periodical cause of anxiety to the diplomacy of
Europe. The recent murder at Bukharest of Professor
Mihaileano, a Macedonian by birth and a Rumanian by
extraction, appears to be a shocking example of the teaching
of a school of political conspirators who have their centre of
operations at Sofia. These persons had already combined to
 blackmail and terrorise the leading Rumanian residents in the
capital of Bulgaria, where the most abominable outrages are
stated to have been committed with impunity. Apparently, they
have now carried the war, with surprising audacity, into the
Rumanian capital itself. Two persons marked out for vengeance
by the terrorists of Sofia had previously been murdered in
Bukharest, according to our Vienna Correspondent, but these
were Bulgarians by birth. It is a further step in this
mischievous propaganda that a Rumanian subject, the occupant
of an official position at the seat of the Rumanian
government, should be done to death by emissaries from the
secret society at Sofia. His crime was that, born of Rumanian
parents in Macedonia, he had the boldness to controvert in the
Press the claims of the Bulgarians to obtain the upper hand in
a Turkish province, where Greeks, Turks, Bulgarians,
Albanians, and Serbs are inextricably mixed up. Professor
Mihaileano had probably very good reasons for coming to the
conclusion that, whatever may be the evils of Ottoman rule,
they are less than those which would follow a free fight in
the Balkans, ending, it may be, in the ascendency of Bulgarian
ruffianism.

"It is for this offence that M. Mihaileano suffered the

penalty of death by the decree of a secret tribunal, and at
the hands of assassins sent out to do their deadly work by
political intriguers who sit in safety at Sofia. The most
serious aspect of the matter, however, is the careless and
almost contemptuous attitude of the Bulgarian Government. The
reign of terror at Sofia and the too successful attempts to
extend it to Rumania have provoked remonstrances not only from
the government at Bukharest, but from some of the Great
Powers, including Austria-Hungary, Germany, and Italy. … There
is only too much reason to fear, even now, that both the
Bulgarian Government and the ruler of the Principality are
afraid to break with the terrorists of Sofia.
{48}
Political assassination is unfortunately among the traditions
of the Bulgarian State, but it has never been practised with
such frequency and impunity as under the rule of Prince
Ferdinand. … His own conduct as a ruler, coupled with the
lamentable decline of the spirit of Bulgarian independence,
which seemed to be vigorous and unflinching before the
kidnapping of Prince Alexander, has steadily lowered his
position. The Bulgarian agitation—to a large extent a sham
one—for the 'redemption,' as it is called, of Macedonia is a
safety-valve that relieves Prince Ferdinand and those who
surround him from much unpleasant criticism. …

"The situation in the Balkans is in many respects disquieting.

The Bulgarian agitation for the absorption of Macedonia is not
discouraged in high quarters. The hostility of the Sofia
conspirators to the Koutzo-Wallachs, the Rumanians of
Macedonia, is due to the fact that the latter, being a small
minority of the population, are ready to take their chance of
equal treatment under Turkish rule, subject to the supervision
of Europe, rather than to be swallowed up in an enlarged
Bulgaria, dominated by the passions that now prevail in the
Principality and that have been cultivated for obvious
reasons. Russia, it is believed, has no wish to see Bulgarian
aspirations realized, and would much rather keep the
Principality in a state of expectant dependence. Servia and
Greece would be as much embarrassed as Rumania by the success
of the Bulgarian propaganda, and Austria-Hungary would regard
it as a grave menace. Of course the Turkish government could
not be expected to acquiesce in what would, in fact, be its
knell of doom. … In Greece, the insubordination in certain
sections of the army is a symptom not very alarming in itself,
but unpleasantly significant of latent discontent. In Turkey,
of course, the recrudescence of the fanaticism which
periodically breaks out in the massacres of the Armenians
cannot be overlooked. A more unfortunate time could not be
chosen for endeavouring to reopen the Eastern question by
pressing forward the Bulgarian claim to Macedonia. Nor could a
more unfortunate method be adopted of presenting that claim than
 that of the terrorists who appear to be sheltered or screened
at Sofia."

London Times, August 23, 1900.

See, also (in this volume),

TURKEY: A. D. 1899-1901.

BALKAN AND DANUBIAN STATES:

Bulgaria.

On the 15th of July, 1895, M. Stambouloff, lately the powerful

chief minister in the Bulgarian government, but now overthrown
and out of favor, was attacked by four assassins, in the
streets of Sofia, and received wounds from which he died three
days afterwards.

The increasing influence of Russia in Bulgaria was manifested

unmistakably on the 14th of February, 1896, when Prince Boris,
the infant son and heir of the reigning Prince Ferdinand, was
solemnly baptised into the Orthodox Greek Church, the Tzar of
Russia, represented by proxy at the ceremony, acting as
sponsor. This is understood to have been done in opposition to
the most earnest remonstrances of the mother of the child, who is
an ardent Roman Catholic, the father being nominally the same.

BALKAN AND DANUBIAN STATES: Montenegro:

Recent changes.

"The accession of territory obtained under the Berlin Treaty

has already begun to alter the character of the country. The
area of the Principality has been almost doubled, and fertile
valleys, tracts of rich woodland and a strip of sea-coast have
been added to the realm of Prince Nikolas. Montenegro is now
something more than the rocky eyrie of a warlike clan, and the
problem of its commercial development constantly occupies the
mind of its ruler. The state of transition is reflected in the
aspect of the capital. A tiny hamlet in 1878, Tzetinye now
bears witness to the growth of civilisation and to the
beneficent influence of a paternal despotism. … Nikolas I.,
'Prince and Gospodar of free Tzrnagora and the Berda,' is the
most picturesque and remarkable figure in the South Slavonic
world. Descended from a long line of heroes, the heir of the
Vladikas, he has, like them, distinguished himself in many a
hard-fought conflict with the hereditary foe. In the field of
poetry he has also won his triumphs; like his father Mirko,
'the Sword of Montenegro,' he has written lyric odes and
ballads; like his ancestor, the Vladika Petar II., he has
composed historical dramas, and his poems and plays hold a
recognised place in contemporary Slavonic literature. The
inheritor of a splendid tradition, a warrior and a bard,
gifted by nature with a fine physique and a commanding
presence, he forms the impersonation and embodiment of all
that appeals most to the imagination of a romantic and
impressionable race, to its martial instinct, its poetic
temperament, and its strange—and to us
incomprehensible—yearning after long-vanished glories. … Any
attempt to describe Prince Nikolas' work as an administrator
and a reformer would lead me too far. The codification of the
law, which was begun by his ancestors, Danilo I. and Petar I.,
has been almost completed under his supervision. … The
suppression of the vendetta is one of the greatest of the
Prince's achievements. … Crime is now rare in the
Principality, except in the frontier districts, where acts of
homicide are regarded as justifiable, and indeed laudable, if
perpetrated in payment of old scores, or if the victim is an
Albanian from over the border. Primary education has been made
universal, schools have arisen in every village, and lecturers
have been appointed to explain to the peasants the advantages
of learning. Communications are being opened up, and the
Principality, which a few years since possessed nothing but
mule-tracks, can now boast of 138 miles of excellent
carriage-road, better engineered and maintained than any I
have seen in the Peninsula. The construction of roads is
 viewed with some apprehension by the more conservative
Montenegrins, who fear that their mountain stronghold may lose
its inaccessible character. But the Prince is determined to
keep abreast of the march of civilisation. Nine post-offices
and thirteen telegraph stations have been established. The
latter, which are much used by the people, will play an
important part in the next mobilization of the Montenegrin
army. Hitherto the forces of the Principality have been called
together by stentorian couriers who shouted from the tops of
the mountains. A great reform, however, still remains to be
attempted—the conversion of a clan of warriors into an
industrial nation. The change has been rendered inevitable by
the enlargement of the bounds of the Principality, and its
necessity is fully recognised by the Prince.
{49}
Once the future of the country is assured, his order will be
'à bas les armes.' He is aware that such an edict would be
intensely unpopular, but he will not flinch when the time for
issuing it arrives. Every Montenegrin has been taught from his
cradle to regard warfare as his sole vocation in life, and to
despise industrial pursuits. The tradition of five hundred
years has remained unbroken, but the Prince will not hesitate
to destroy it. So enormous is his influence over the people,
that he feels confident in his ability to carry out this
sweeping reform."

J. D. Bourchier,
Montenegro and her Prince
(Fortnightly Review, December, 1898).

BALKAN AND DANUBIAN STATES: Montenegro:

New title of the Prince.

On the 19th of December, 1900, at Tzetinye, or Cettigne, "the

President of the Council of State, in the presence of the
other Ministers and dignitaries and of the members of the
Diplomatic Corps, presented an address to the Prince of
 Montenegro praying him, in token of the gratitude of the
Montenegrin people for the benefits which he had conferred on
them during his 40 years' reign, to take the title of Royal
Highness. The Prince acceded to the request, and, replying to
the President, thanked all the European rulers who on this
occasion had given him a fresh proof of their friendship by
their recognition of his new title. After the ceremony a Te
Deum was celebrated in the Cathedral, and the Prince
subsequently reviewed the troops, receiving a great welcome
from the people."

Telegram,
Reuter's Agency.

BALKAN AND DANUBIAN STATES:

Servia.

In January, 1894, the young king, Alexander, called his

father, the ex-king, Milan (abdicated in 1889—see, in volume
1. BALKAN AND DANUBIAN STATES: A. D. 1879-1889), to Belgrade
to give him help against his Radical ministers, who had been
taking, the latter thought, too much into their own hands. The
first result was a change of ministry, soon followed by a
decision from the synod of Servian bishops annulling the
divorce of ex-King Milan and Queen Natalie; by a public
announcement of their reconciliation, and by an ukase from
King Alexander, cancelling all laws and resolutions which
touched his parents and restoring to them their rights and
privileges as members of the royal house. This, again, was
followed, on the 21st of May, by a royal proclamation which
abolished the constitution of December, 1888, and restored the
old constitution of 1869. This was a tremendous step backward, to
a state of things in which almost no protection against
arbitrary kingship could be found.

For some years the ex-king exercised considerable influence

over his son, and was again an uncertain and much distrusted
 factor in the troubled politics of southeastern Europe. In
1898 the son appointed him commander-in-chief of the Servian
army, and he is said to have ably and energetically improved
its efficiency during the brief period of his command. A
breach between father and son was brought about before long,
however, by the determination of the latter to marry a lady,
Madame Draga Maschin, considerably older than himself, who had
been lady-in-waiting to his mother; while the father was
arranging a political marriage for him with a German princess.
The young king married his chosen bride in August, 1900, and
guarded his frontier with troops to bar the return of his
father, then sojourning at a German watering place, to the
kingdom. It was a final exile for the ex-king. He visited
Paris for a time; then went to Vienna, and there, on the 11th
of February, 1901, he died, at the age of 47.

BALLOONS, Declaration against explosives from.

See (in this volume)

PEACE CONFERENCE.

BALTIC and NORTH SEA CANALS.

See (in this volume)

GERMANY: A. D. 1895 (JUNE); and 1900 (JUNE).

BANK OF FRANCE: Renewal of privileges.

See (in this volume)

MONETARY QUESTIONS: A. D. 1897.

BANKING: Its effect on the Nineteenth Century.

See (in this volume)

NINETEENTH CENTURY: THE TREND.

BANKRUPTCY LAW, National.

 See (in this volume)
UNITED STATES OF AMERICA: A. D. 1898 (JULY 1).

BARBADOS: Condition and relief measures.

See (in this volume)

WEST INDIES, THE BRITISH: A. D. 1897.

BARCELONA: A. D. 1895.
Student riots.

See (in this volume)

SPAIN: A. D. 1895-1896.

BAROTSILAND:
British Protectorate proclaimed.

See (in this volume)

SOUTH AFRICA (RHODESIA): A. D. 1900 (SEPTEMBER).

BARRAGE WORKS, Nile.

See (in this volume)

EGYPT: A. D. 1898-1901.

BARRIOS, President: Assassination.

See (in this volume)

CENTRAL AMERICA (GUATEMALA): A. D. 1897-1898.

BARTON, Miss Clara, and the Red Cross Society.

Relief work in Armenia and Cuba.

See (in this volume)

ARMENIA: A. D. 1896 (JANUARY-MARCH);
and CUBA: A. D. 1896-1897.
BASHEE,
BACHI ISLANDS, The American acquisition of.

See (in this volume)

UNITED STATES OF AMERICA: A. D. 1898 (JULY-DECEMBER).

BECHUANALAND, British:
Annexation to Cape Colony.

See (in this volume)

AFRICA: A. D. 1895 (CAPE COLONY).

BECHUANALAND, British:
Partial conveyance to the British South Africa Company.

See (in this volume)

AFRICA: A. D. 1895 (BECHUANALAND).

BEEF INVESTIGATION, The American Army.

See (in this volume)

UNITED STATES OF AMERICA: A. D. 1898-1899.

BEET SUGAR.

See (in this volume)

GERMANY: A. D. 1896 (MAY);
and SUGAR BOUNTIES.

BEHRING SEA.

See (in this volume)

BERING SEA.

BÊL, Temple of:

Exploration of its ruins at Nippur.
 See (in this volume)
ARCHÆOLOGICAL RESEARCH: BABYLONIA: AMERICAN
EXPLORATION.

BELGIAN ANTARCTIC EXPEDITION.

See (in this volume)

POLAR EXPLORATION, 1897-1899.

{50}

BELGIUM: A. D. 1894-1895.
The first election under the new constitution.
Victory of the Catholics and surprising Socialist gains.

Elsewhere in this work the full text of the Belgian

constitution as it was revised in 1893;

See in volume 1
CONSTITUTION OF BELGIUM).

The peculiar features of the new constitution, especially in

its provision of a system of cumulative or plural voting, are
described.

See in volume 3
NETHERLANDS (BELGIUM): A. D. 1892-1893)

The singularity of the experiment thus introduced caused the

elections that were held in Belgium in 1894 and 1895 to be
watched with an interest widely felt. Elections for the
Chamber of Representatives and the Senate occurred on the same
day, October 14, 1894. Previously the Belgian suffrage had
been limited to about 130,000 electors. Under the new
constitution the electors numbered no less than 1,370,000, and
the working of the plural system gave them 2, 111,000 votes.