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Application of fluorescence lifetime imaging(FLIM) in latent finger mark detection
L.K. Seah *, P. Wang, V.M. Murukeshan, Z.X. Chao
School of Mechanical and Aerospace Engineering, Nanyang Technological University,
50 Nanyang Avenue, Singapore 639798, Singapore
Received 14 April 2005; received in revised form 1 August 2005; accepted 24 August 2005
Available online 22 September 2005
Abstract
Fluorescence lifetime imaging (FLIM) in frequency domain enables the mapping of the spatial distribution of fluorescence
lifetimes of a specimen. It has been extensively applied in biology. In this paper, a theoretical analysis for the fluorescence
lifetime determination of latent finger mark samples is described, which is followed by the feasibility study of using FLIM in
frequency domain for latent finger marks detection. Preliminary experiments are carried out with latent finger marks treated with
a fluorescent powder on two different substrates. The resulting fluorescence lifetime image of finger mark revealed a good
contrast, and was able to detect the latent finger marks clearly.
# 2005 Elsevier Ireland Ltd. All rights reserved.
Keywords: Fluorescence lifetime imaging; Frequency domain; Latent finger mark; Homodyne
1. Introduction
Finger marks are one of the most valuable types of
physical evidence and hence its detection plays a significant
role in criminal investigation and forensic science. There are
three types of finger marks that occur at a crime scene:
visible, plastic and latent. Latent finger mark is the most
difficult to be detected. The existing detection techniques for
latent finger marks have their own limitations. Most con-
ventional detection methods (physical and chemical) cannot
detect older marks [1,2]. Laser induced fluorescence detec-
tion of latent finger mark was initially explored in 1976 and
involves the detection of fluorescence intensity, color and
lifetime [9]. In general, conventional methods for detectionof latent finger marks take the first two properties into
consideration. However, the existing techniques based on
fluorescence filtering fail when there is strong fluorescence
emission from the background, and are ineffective when the
emission wavelengths from the finger mark and that of
background fall in the close wavelength range. In this
context, FLIM method would be beneficial.
Fluorescence lifetime is the average decay time of the
fluorescence emitted by a molecule after excitation with a
short laser pulse. Fluorescence lifetime imaging (FLIM)
has received considerable attention and has been widely
used in biophysics and medical diagnosis [36]. FLIM
could obtain the unique and quantitative information
available from dynamic fluorescence measurements, and
possibly distinguish several fluorescence species with
dissimilar lifetimes even though they may have overlap-
ping spectra.
This paper deals with determination of average fluores-cence lifetimes of latent finger mark sample on a pixel-by-
pixel basis using homodyne detection method. A number of
images of the sample are collected at different phase delays
relative to the excitation light. A subsequent fit of fluores-
cence intensity on a pixel-by-pixel basis yields the fluores-
cence lifetime distribution image of the latent finger mark,
which is independent of the fluorescence intensity.
www.elsevier.com/locate/forsciintForensic Science International 160 (2006) 109114
* Corresponding author. Tel.: +65 6790 4824; fax: +65 6795 4632.
E-mail address: [email protected] (L.K. Seah).
0379-0738/$ see front matter # 2005 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.forsciint.2005.08.018
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2. Principle and theory of fluorescence lifetime
measurement
2.1. Fluorescence lifetime measurement in frequency
domain
The measurement of fluorescence lifetime is based on the
cross-correlation method introduced by Spencer and Weber
[7] (Fig. 1). The lifetime resolved fluorescence experiment is
carried out by modulating the excitation light sinusoidally at
a high frequency (typically on order of 10100 MHz) and
determining the demodulation and phase of high frequency
modulated fluorescence emission.
The fluorescence lifetime can be calculated by measuring
the phase shift and demodulation of the fluorescence emis-
sion relative to the phase and demodulation depth of the
excitation light.
If the excitation light intensity is modulated as a sinu-
soidal function of time Ev(t) with a frequency f, the fluor-escence emission intensity is a sinusoidal function with the
same frequency [8]:
Evt E0;v1 ME;vsinv t fE;v (1)
Fvt F0;v1 MF;vsinv t fF;v: (2)
where E0,v is the time-independent offset, ME,v the relative
modulation of the excitation light, v the radial frequency
(v = 2pf)andfE,v is the arbitrary phase lag of the excitation.
The symbols F0,v, M0,v, MF,v and fF,v of Eq. (2) are similar
but refer to the fluorescence emission. The total phase lag of
the fluorescence emission relative to the excitation light Df
and modulation Mcan be expressed by
Df fF;v fE;v (3)
MMF;v
ME;v(4)
For a single lifetime, the lifetime can be determined from
both Df and M:
tf 1
vtanDf (5)
tM1
v
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi1
M2 1
r(6)
where tf and tMare the lifetimes calculated from Df and M,
respectively. If they aredifferent, it can be concluded that the
system is heterogeneous. In this paper, the average lifetime
was calculated by Eq. (5).
The appropriate modulation frequencies for measuring
fluorescence lifetimes, which fall in the range of 110 ns, are
20100 MHz. It is impossible to directly measure the phase
and demodulation of the time-varying signals with such high
frequency. Therefore, the high frequency signal is trans-
formed to either DC (homodyne measurement) or very low
frequencies (heterodyne measurement), hence the phase and
demodulation can easily be measured. In this paper, homo-
dyne method was used as it is relatively simple compared to
heterodyne method.
2.2. Homodyne detection
The principle of the homodyne measurement can be
introduced easily for the simplest case of a perfectly sinu-soidal excitation and a single fluorescence lifetime. For
homodyne detection, the source and detector frequencies
are the same. The intensity is proportional to the sine
function of the phase difference between the detector and
the emission. To acquire the entire phase and modulation
information, it is necessary to change the phase of the
detecting device relative to that of the excitation light. In
most of the case, the gain of the detection is modulated
according to
Gvt G0;v1 MG;vsinv t fG;v: (7)
where G0,v is the time-independent offset, MG,v the relative
modulation of the gain and fG,v is the arbitrary phase lag ofthe gain.
Then the detected intensity is given by
F0t F0;01 MF;0sinf0G;v fF;v; (8)
where F0;0 F0;vG0;v; MF;0 12MF;vMG;v andf
0G;v
fG;v p2:
To obtain Df and M, a reference measurement must be
made by measuring a reference compound with known
fluorescence lifetime (tR), the detected signal has the similar
expression
R0t R0;01 MR;0sinf0G;v fR;v (9)
where R0;0 R0;vG0;v andMR;0 12 MR;vMG;v:Then Df and Mare defined as
Df fF;v fs;v fF;v fR;v tan1vtR (10)
MMF;v
MS;v
MF;v
MR;v
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi1 vtR
2q (11)
The subscript Sdenotes scattered light whose effective
lifetime is zero.
L.K. Seah et al. / Forensic Science International 160 (2006) 109114110
Fig. 1. Phase shift Df and demodulation Mof the fluorescence
signal with respect to the excitation light. The modulation
M= (ACem/DCem)/(ACex/DCex).
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2.3. Data analysis
The data are taken as a series of subimages collected at
equidistant phase steps. For Eqs. (10) and (11), the key is to
get the value ofMF,v and fF,v. For a convenient analysis,
Eq. (8) is redefined as:
yt;i a11 a2sinxi a3 (12)
where a1 = F0,0, a2 = MF,0, a3 = fF,v and xi f0G;v;i,
where f0G;v;i is the different phase value of the gain. The
goal of curve fitting is to find the value ofa1, a2 and a3 that
will make the calculated results yt,i closest to the detected
signals yd,i [8]. Eq. (12) is a non-linear relationship, but a
linear expression could be obtained through transformation:
yt;i c1 sin xi c2 cos xi c3 (13)
where c1 a1a2 cos a3; c2 a1a2 sin a3 and c3 a1:In order to do the linear curve fit, Eq. (13) was regarded as
multi-variable function. The images include the informationofyd,i and xi. After the process of multi-variable curve fit, the
values of coefficients c1, c2 and c3, are obtained. The values
ofa1, a2 and a3, are calculated by
a1 c3
a2
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffic21 c
22
pc3
a3 tanc2
c1
b1 > 0
a3 tanc2
c1
p b1 < 0
8>>>:
(14)
Through the same process, a2,R and a3,R are calculated.
The Mand Df are obtained by simple calculation:
Ma2;F
a2;R
1ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi1 vtR
2q (15)
Df a3;F a3;R tan1vtR; (16)
where Fand R refer to the sample and the reference signal,
respectively.
3. Experimental setup and procedure
The schematic diagram of the experimental set up is
shown in Fig. 2. A CW Argon laser (Coherent Innova 90 C)
is modulated using an electro optic modulator (Conoptics
EOM, Model 350-210) capable of operating in the range of
0100 MHz. A power amplifier is used as the power source
to the modulator, which is modulated by a sinusoidal signal
output from function generator 1 (Agilent, Model 33250A).
The modulated laser light is coupled into a single mode
optical fiber (OZ optics) to illuminate the sample treated
with fluorescence powders. An intensified charge coupled
device camera (ICCD, Lavison, Pico Star HR) collects the
resulting fluorescence emission from the sample. Function
generator 2 (Agilent, Model 33250A) is used as the signalsource to modulate the gain of ICCD. The two function
generators aresynchronized. A long pass optical filter is used
to cut off the excitation-scattered light from the sample. The
ICCD is connected with a camera interface board and
controlled by a computer.
By changing the phase of the function generator 2, the
phase shift of the detecting device relative to the exciting
light is changed. In order to have a good fit, 10 images are
obtained in one period equal spaced out of 368 Then the
different phase step of the gain is defined as:
X 0;p=5; 2p=5; 3p=5; 4p=5;p; 6p=5; 7p=5; 8p=5; 9p=5
After 10 sample images and 10 reference images are
collected at equidistant phase steps, they are used to calcu-
late the lifetime by FLIM program, which is implemented in
MATLAB. The program is outlined schematically in Fig. 3.
First a2,R and a3,R are obtained by calculating the reference
data. After importing the source data, for every pixel whose
L.K. Seah et al. / Forensic Science International 160 (2006) 109114 111
Fig. 2. Schematic diagram of the experiment setup.
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intensity is within the threshold, the intensity values of
equidistant phase steps are fitted to a sine wave to yield
the demodulation factor a2,Fand the phase shift factor a3,F.
In order to process the curve fitting procedure, Eq. (13) is
transformed to
Y c1
sinX c2
cosX c3
(17)
The function in MATLAB toolbox is used to obtain the
coefficients. Eq. (14) is used to obtain the value ofa1,F, a2,F
and a3,F. Then MandDf arecalculated to decide the lifetime
through Eqs. (15) and (16). After every pixel is processed,
the maximum and minimum values are set as the value range
to output the lifetime image.
In order to simulate a condition of having very close
emission wavelengths for finger mark and background
fluorescence, fresh finger marks from a single person are
deposited on fluorescing color paper and treated with blitz-
green powder (Lightning Powder Company) that is applied
L.K. Seah et al. / Forensic Science International 160 (2006) 109114112
Fig. 3. Block diagram of FLIM program.
Fig. 4. The intensity image (a) and fluorescence lifetime image (b) of the blitz-green-treated finger mark samples on green fluorescence paper
substrate.
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by means of a magneticbrush.The emissionwavelength of the
fluorescing color paper is 516 nm and its lifetime is 3.1 ns,
while those of blitz-green powder are 523 nm and 8.96 nm.
Experiment was also carried out on the finger mark
treated with blitz-green fluorescent powder on smooth
calendar paper. The background has two major fluores-
cence components with lifetime value 13.42 ns and
2.56 ns. Lifetime value of finger mark sample treated
with blitz-green powder is 8.96 ns, which is shorter than
that of the major fluorescence component (13.42 ns) from
the background
4. Result and discussion
Selecting a 700 700 pixel region, the intensity imageof the finger mark on green fluorescence paper treated
with blitz-green powder is shown in Fig. 4a. The contrast
of the latent finger mark image is found to be poor and
hence could not identify the ridge details. This is due to
that the emission wavelength of green fluorescence paper
(516 nm) is merged with that of the latent finger mark
treated with blitz-green fluorescent powder (523 nm).
Fig. 4b shows the corresponding average lifetime image
of latent finger mark. The fluorescence powder has longer
lifetime than the background fluorescence of the green
paper. It can be seen that lifetime image effectivelysuppress the background fluorescence so as to give a clear
ridge details of the finger mark
Fig. 5 shows the intensity image and fluorescence life-
time image of the finger mark treated with blitz-green
powder on postcard. Due to the strong multiple fluorescence
emissions from the background, the latent finger mark
cannot be identified under normal imaging. As mentioned
in the previous case, in the fluorescence lifetime image the
background can be subdued successfully despite the back-
ground fluorescence is having a higher intensity emission.
For the case that the background has longer lifetime, a
negative image will be obtained compared to Figs. 4b and 5b.
The finger mark can also be detected in that case.
5. Conclusion
FLIM has been widely used in biology and biomedical
fields. In this paper, lifetime imaging of latent finger marks
on two different substrates were carried out. An approach
that involved the calculation of total phase lag and demo-
dulation factor is used to determine the lifetimes pixel by
pixel. The results show that the FLIM can detect the latent
finger mark whose emission wavelength falls in the close
wavelength range with that of the background. The ridge
details of latent finger mark can be identified clearly. The use
of lifetime imaging yields results that are independent of the
fluorescence intensity. FLIM technique is a general approach
for latent finger mark detection and is only limited by the
lifetime difference between the substrate and the finger
mark. Hence, as long as there is a lifetime difference that
can be detected, this technique can be applied.
Acknowledgements
The authors gratefully acknowledge the support and fund-ing from The Enterprise Challenge (TEC), Singapore and
Academic Research Fund (AcRF), Nanyang Technological
University. The authors express their sincere gratitude to
Singapore Police Force for their collaboration in this project.
References
[1] E.R. Menzel, Fingerprint Detection with Lasers, second ed.,
Marcel Dekker, New York, 1999.
L.K. Seah et al. / Forensic Science International 160 (2006) 109114 113
Fig. 5. The intensity image (a) and fluorescence lifetime image (b) of the blitz-green-treated finger mark samples on postcard substrate.
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[2] H.C. Lee, R.E. Gaensslen, Advances in Fingerprint Technology,
Elsevier, New York, 1991.
[3] R. Sanders, H.C. Gererritsen, Fluorescence lifetime imaging of
free calcium in single cells, Bioimaging 2 (1994) 131
138.
[4] D. Elson, S. Webb, J. Siegel, K. Suhling, D. Davis, J. Lever, D.Phillips, A. Wallace, P. French, Biomedical applications of
fluorescence lifetime imaging, Opt. Photon. News 11 (2002)
2732.
[5] K. Dowling, M.J. Dayel, Hyde SCW, French PMW, M.J. Lever,
J.D. Hares, High resolution time-domain fluorescence lifetime
imaging for biomedical applications, J. Mod. Opt. 46 (2) (1999)
199209.
[6] M. Elangovan, R.N. Day, A. Periasamy, Nanosecond fluores-
cence resonance energy transfer-fluorescence lifetime imaging
microscopy to localize the protein interactions in a single living
cell, J. Microsc. 205 (2002) 314.
[7] R.D. Spencer, G. Weber, Measurements of subnanosecond
fluorescence lifetime with a cross-correlation phase fluorometer,Ann. N. Y. Acad. Sci. 158 (1969) 361376.
[8] Gadella TWJ, R.M. Clegg, T.M. Jovin, Fluorescence lifetime
imaging microscopy pixel-by-pixel analysis of phase-modula-
tion data, Bioimaging 2 (1994) 135159.
[9] L.K. Seah, U.S. Dinish, S.K. Ong, Z.X. Chao, V.M. Muruke-
shan, Time-resolved imaging of latent fingermarks with nano-
second resolution, Opt. Laser Tech. 36 (5) (2004) 371376.
L.K. Seah et al. / Forensic Science International 160 (2006) 109114114