Optics and Photonics Journal, 2011, 1, 197-203
doi:10.4236/opj.2011.14031 Published Online December 2011 (http://www.SciRP.org/journal/opj)
Real Time Monitoring of Fluorescent Particles in
Micro-Channels by High Resolution Dual Modality Probe
Imaging
Krishnan Sathiyamoorthy, Valiyambath Krishnan Mohankumar, Vadakke Matham Murukeshan
School of Mechanical and Aerospace engineering, Nanyang Technological University, Singapore
Received July 27, 2011; revised September 6, 2011; accepted September 26, 2011
Abstract
Imaging of micro particles in micro-fluidic channels is one of the recent thrust areas in research as it pro-
vides more flexibility in setup and analysis compared to conventional microscopy. However, a probe based
imaging scheme, with achievable high resolutions incorporating multimodal analysis is one of the challenges
researchers have been facing. In this context, this paper illustrates a simple dual modality high resolution
flexible probe imaging system for imaging applications in micro/optofluidic channels. The proposed system
exhibits axial and lateral resolution of about 16 μm and 3.12 μm respectively. This proposed system also ex-
hibits a modulation transfer function (MTF) of about 38.42%. The performance of the system is validated by
imaging micro particles in a microchannel and obtaining the fluorescent emission spectrum simultaneously.
Keywords: Microscopy, Image Fiber, Spectrophotometer, Microchannel
1. Introduction
Micro-fluidic devices have found an exquisite position in
the biomedical technology in recent decades due to their
successful implementation in the development of DNA
chips, lab-on-a-chip and optofluidic technologies [1-9].
Micro-channels have been implemented as pseudo blood
vessels with confocal Particle Image Velocimetry (mi-
cro-PIV) as the detection system to better understand
blood rheology [4,7]. Microchannels also find applica-
tion in biochemical analysis as they help in reducing the
usage of analytes significantly. Moreover, a complete
microchemical system with a large number of micro-
channels for simultaneous analysis of a large number of
samples becomes possible as a result of its miniature size
and easy fabrication [4]. The detection modes in these
systems can be as varied as UV-vis absorbance, refrac-
tive index measurements, electrochemical detection, or
laser-induced fluorescence detection. But most of the
detection systems remained aloof from the microchannel
as close monitoring was found to be less feasible. Hence
the development of on-chip methods for the investigation
of physical, chemical and biological processes is receiv-
ing rapidly increasing attention [3,5,6]. Image fiber
based probe system will help in interrogation of remote
sites due to its flexibility and miniature size [10-15].
Several groups have investigated different configurations
of fiber based imaging systems for diverse applications
[11,14-22]. For example, the flexibility miniature size of
the image fiber based optical imaging system enables it
to be readily integrated with commercially available en-
doscopic systems.
Fiber-based profilometer is preferred in inspecting hard-
to-reach surfaces than microscope-type profilometers
because of their size. Confocal Particle Image Velocime-
try (micro-PIV) has been successfully employed in mi-
crochannel to study the blood rheology [4,7]. But its
flexibility and ease of application in a system containing
a large number of microchannels for multianalyte analy-
sis is curtailed due to its bulky size. Flexible fiber image
guides can be used to transmit spatially parallel optical
data between a multichannel array in large microchannel
system and a photodetector array [23].
Besides, image fiber based system has an advantage
that it can also be operated in a quasi/confocal regime due
to possible excitation of each individual fiberlets/group
of fiberlets. It can also enable imaging of the targeted
sample without mechanical scanning by adopting point
by point illumination of individual fiberlets at a time
employing appropriate optics at the appropriate planes
[11-15,19,24].

K. SATHIYAMOORTHY ET AL.
198
In this context, this paper focuses on the feasibility of
a fiber based high resolution image system to image and
probe any activity that takes place in the microchannels.
The proposed system is employed as a dual modality
system and is validated by imaging microspheres in a mi-
crochannel and simultaneously recording their fluores-
cence emission.
2. System
The schematic diagram of the proposed experimental
set-up is shown in Figure 1. A solid state laser operating
at the wavelength of 532 nm is employed as the source
beam. It is focused on to the proximal end of the image
fiber using 60×/1.20 microscope objective lens (OLY-
MPUS UPLANSAPO 60×/1.20 objective). The image
fiber (Sumitomo Electric, Japan) used here encapsulates
about 50000 fiberlets (pixel fiber). The size of the image
fiber is 1 mm in diameter. The core diameter of each
pixel fiber and distance between neighboring cores are
2.7 μm and 4.4 μm respectively.
At the distal end, the light guided through the image
fiber is focused on the sample by the custom made mi-
niature objective lens which offers small size and favor-
able optical geometry with plane optical surfaces. The
size of the objective lens is almost same as the size of the
image fiber and has a working distance of 0.3 mm.
The light from the sample is guided back through the
image fiber by the same objective lens, it is then allowed
to pass through the 60× objective lens and finally di-
rected towards CCD camera (Basler Scout SCA1400fc
model). The camera offers the resolution 1392 × 1040
pixels (each pixel has size 6.45 × 6.45 μm) with speed of
30 frames per second.
The system is automated using NI image acquisition
system (IEEE 1394b). The backscattered lights from the
sample and from other locations are filtered by high pass
filter and allow only the fluorescent emission entering
the camera. The choice of the filter depends on excitation
and emission wavelength of the test sample. High pass
filters of various cut-off wavelengths can be employed
according to the requirements.
Figure 1. Schematic diagram of the proposed system.
The beam splitter, objective lens and fiber bundle con-
stitute the intermediate optical component unit of probe
image system. A 50/50 pellicle beamsplitter, which is
mostly immune to ghost images that arise from endface
reflections of the beam splitter, is used to direct light
from source to fiber bundle and redirect the light from
the object to the detector. The CCD camera with filter
forms the detector part of the proposed system. The filter
is used to prevent stray lights, such as reflected, scattered
and diffracted laser lights from various optical elements
of the system, entering the CCD.
2.1 System Capability Analysis
2.1.1 Axi al Resoluti o n
Resolution has been defined as the minimum separation
necessary between two features in a specimen for each to
be seen as two separate objects in the image.
Full width half maximum (FWHM) of intensity profile
is estimated using the Gaussian model




 

2
0π/2exp 2/ c
yAW xxW  (1)
where A is the intensity at the centre of the beam at its
waist, W is the radius at which the intensity drop to 1/e2
of their axial value and (x-xc) is the radial distance from
the center axis of the beam.
The measurement of depth response is performed by
using technique proposed by Hamilton et al. [17,25]. The
axial resolution of the system is measured by placing a
plane mirror (λ/20) at the object plane and the mirror is
moved/ scanned towards the system through the effective
focal plane of the system. [17,24,25] The light reflected
back to the system is monitored by a photodiode kept at
the image plane
Figure 2 represents the intensity measured as a func-
tion of distance. The measured FWHM is found to be
15.98 ± 0.94 μm.
Figure 2. Measurements of axial resolution of the proposed
system.

199 K. SATHIYAMOORTHY ET AL.
2.1.2. Axi al R e sol ution
Multi-bar test charts are most often used to determine the
overall resolution limit of an entire optical system as
each component of a system adds some degradation to
image quality. Hence the effective lateral resolution of
the system is measured by imaging U.S. Air Force bar
target (USAF1951 chart) [26]. White light is used for
this measurement. The system is able to image the high-
est spatial frequency (group 7) labeled on the USAF
chart. The Figures 3 (a) and (b) represent the images of
two highest spatial frequencies (group 6 and 7) of USAF
chart recorded by using the proposed system. The circle
which is marked around element 2 of group 7 in Figure
3 (b) represents the maximum resolution limit achievable
by the system.
Calculation indicates that the present system has a lat-
eral resolution of 16 μm and 3.12 μm. The colour of the
image is due to usage of the filter (Chroma) at the cam-
era end.
The contrast of the system can be determined from the
edge resolution which can be obtained by measuring the
linear reflected intensity profile across the edge of any
one of the bar pattern in USAF chart.
The measurement of contrast ratio is important as it
gives the system ability to distinguish the target signal
from the background noise (e.g. background signal in the
tissue) to obtain well defined image. It can be calculated
with respect to highest spatial frequency the system can
able to resolve. Figure 4 represents the linear reflected
intensity profile obtained across the element 2 group
seven of USAF chart. For normal operating conditions,
the contrast ratio should be greater than 50%. The equa-
tion to find modulation transfer function (MTF)


()100% *0MTF fCfC (2)
where
 
0WB WB CVVVV 
and
 
max minmax min
Cf V VV V ,
VB and VW—The average luminances of black and white
(a) (b)
Figure 3. Measurement of lateral resolution using USAF
(1951) chart.
Figure 4. Measurement of lateral resolution by means of
linear intensity profile obtained across the element 2 of
group 7 of USAF chart.
areas of low spatial frequencies bars (group 1) of USAF-
1951 chart respectively and Vmin and Vmax -the minimum
(negative peak) and the maximum (positive peak) lumi-
nances for a pattern of maximum resolvable spatial fre-
quency of the current system (in the present case it is 2nd
element of group 7).
The proposed system exhibit the MTF of about 38.42%.
3. Imaging and Spectroscopic Analysis of
Fluorescent Particles in Microchannel
The microchannel is prepared in translucent ‘Topaz’
(Figure 5(a)) which is a thermoplastic olefin polymer of
amorphous structure (COC－Cyclic Olefin Copolymer).
It was molded on a Battenfeld HM25/60 injection mold-
ing machine. The channel is meandering with lengths of
about 6.5 mm and 1.2mm along X and Y directions re-
spectively and has 25 bends. Figure 5 (b) represents the
schematic diagram of the microchannel. The depth and
width of the microchannel are about 38 µm and 90 µm
respectively.
4. Results and Discussion
The Figure 6 represents the image of a single sphere in
the microchannel. The polystyrene sphere used here is a
fluorescent sphere procured from Invitrogen and has an
average size of ≈1 µm. It is carboxylate-modified poly-
styrene microsphere coated with Nile red fluorescent
material which has an emission peak at 641 nm for 532
nm excitation.
Hence band pass filter (Emission 610/75 nm) is inte-
grated at the front facet of the CCD. Microsphere which

K. SATHIYAMOORTHY ET AL.
200
(a) (b)
Figure 5. Microchannel (a) photographic image and (b) schematic diagram of the microchannel.
(a) (b)
(c) (d)
Figure 6. Image of the microparticle in microchannel obtained by the proposed system (a) Image obtained by illuminating the
whole area of image fiber, (b) FFT processed image of (a), (c) The same Image obtained by exciting only a few fiberlets of
the image fiber, and(d) FFT processed image of (c ).
is prepared in ethanol is made to flow in the microchan-
nel and the image is recorded using the described high
resolution fiber probe system.
Figure 6(a) represents the captured image by illumi-
nating the whole area of image fiber. The image is rep-
resented by an array of pixels which collect and transmit
light from a fluorescent sphere located close to the mi-
crochannel edge. The grid-like appearance is due to the
fiberlets of the image fiber. The grid-like appearance can
be a nuisance in contouring the target and can be filtered
out by image processing technique involving FFT trans-
form with subsequent low pass filtering (to remove the
high frequency grid structure elements), followed by
inverse Fourier transform and contrast enhancement.
This process helps in obtaining the image devoid of
grid-like appearance. Figure 6(b) represents the proc-
essed image of Figure 6(a). Figure 6(c) is the image
obtained by exciting only a few fiberlets of the image
fiber (assumed to be operating in the quasi-confocal re-
gime) and Figure 6(d) represents the processed image.
The quality of the image is found to be quite good after
digital processing.

201 K. SATHIYAMOORTHY ET AL.
Fiber based Ocean optics spectrometer is integrated to
the system for the simultaneous spectral analysis of the
sample.
Figure 7 represents the fluorescence spectrum of the
sample flowing through the microchannel.
The recorded spectrum contains an intense narrow ex-
citation peak at 532 nm, followed by broad fluorescence
peak at around 641 nm.
Figure 8 represents the measurement (in terms of peak
fluorescent emission) of liquid flow with respect to time.
The liquid is made to flow at different intervals in the
microchannel and corresponding fluorescence emission
is monitored.
The broad pulse represents the liquid flow with the
fluorescent particles in the microchannel and the duration
of flow of the fluorescent particles is represented by the
width of the pulse. Fiber based image system has been
successfully employed in biomedical imaging systems.
Figure 7. Fluorescence emission of the flow inside micro-
channel due to 532 nm excitation.
Figure 8. Measurement of fluorescence emission at 641 nm
with respect to time.
Different optical configurations suitable for endoscopic
application were proposed by several researchers
[12,14,18]. Knittle et al have developed a miniaturized
confocal laser scanning microscope suitable for diagnostic
endoscopy [12]. They proposed a system that contains no
active mechanical or electronic parts on the endoscopic
head for scanning hence the system will be readily
available for integration with conventional endoscopic
system. 2D scanning was achieved by exciting each in-
dividual fiber of the image fiber by the mechanical scan-
ner at the frontal end. Similarly, our system is very sim-
ple that contains no active elements at the fiber distal and
frontal ends. No mechanical scanning is needed as the
system will be operated in wide field mode. The system
exhibits a simple configuration of CCD camera at the
frontal end and found to be much simpler and cost effec-
tive than the earlier discussed technique. The system can
also be operated in confocal or quasi-confocal regime by
having mechanical optical scanner at the frontal end.
Further, though the image fiber systems have been
used extensively for bio-imaging application, not much
works have been reported towards micro channel appli-
cations. Fiber based system can offer greater flexibility
than conventional system in accessing remote sites. The
application of single fiber for this kind of studies have
been reported earlier but it required different configura-
tions such as fiber tip scanning to acquire full image. In
the current study we explored the possibilities of em-
ploying the image fiber based system for dual-modality
analysis by recording optical spectrum as well as imag-
ing simultaneously.
5. Conclusions
The image fiber based high resolution probe system for
the dual modality imaging of micro/optofluidic channel
is investigated and illustrated in this paper. The devel-
oped system exhibits axial and lateral resolutions of
about 16 μm and 3.12 μm, respectively. The employment
of image fiber offers more flexibility in integrating this
dual modality system with microchannel. The proposed
concepts and probe system can be expected to find ap-
plications in biotechnology, chemical synthesis, analyti-
cal chemistry and optofluidic technologies. The system
can also be operated in confocal/quasi-confocal regime
(which provides better imaging than most of the avail-
able conventional system) by exciting each individual
fiberlet/group of fiberlets of the image fiber.
6. Acknowledgements
The authors acknowledge the financial support received
through ARC 3/08.

K. SATHIYAMOORTHY ET AL.
202
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