Real Time Monitoring of Fluorescent Particles in Micro-Channels by High Resolution Dual Modality Probe Imaging

doi:10.4236/opj.2011.14031

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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

E-mail: [email protected]

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











0π/2exp 2/

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)

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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