WEDNESDAY ,4FTERNOON / CLE0'98 / 307

the reflectance and fluorescence. The probe 5. Ingrams et al., Head & Neck, 19, 27-32
consists of 46 optical fibers in two concentric (1997).
bundles. The center bundle contains 25 fluo-
rescence excitation fibers and 12 fluorescence
collection fibers. At the distal end of the probe, CW03 5 1 5 pm
these fibers are arranged randomly in a central
bundle and placed in contact with a short piece Raman detection of human macular
of thick quartz fiber. Nine fibers for illumina- carotenoid pigments
tion and collection of diffuse reflectance are
W. Gellermann, R. McClane, N. Balashov,*
arranged in a ring around the fluorescence
measurement fiber. The collection fibers are P.S. Bei-nstein,* Department of Physics and 0 1 I
D k o n Laser Institute, University of Utah, Salt 0 80 160 240
located 180°, 90°, and 45' from the illumina-
Lake City, Utah 84112 Carotenoid Content in 5 mm Macular Sample I ng
tion fiber. White light from the Xe lamp is
coupled to the proximal end of a single illumi- We have used resonance Raman scattering as a CWO3 Fig. 2. Intensity of carotenoid 1524
nation fiber. The distal ends of these fibers are novel optical technique to measure the con- cm-' Stokes Raman line vs. carotenoid concen-
flush with the tip of the central fiber and placed centration of carotenoid pigments in the hu- tration as determined by HPLC. Illumination
in contact with the sample surface. Measure- man retina. Using argon laser excitation we conditions: 4880 A, 2 mW, 4 mm spot size, 10 s.
ment requires 3 min for fluorescence and 2 obtained two strong carotenoid resonance Ra- Plot demonstrates linear correlation between the
min for reflectance. man signals from human retinal tissues at 1159 two methods. Correlation coefficient R = 0.94.
Patients with abnormal oral lesions were and 1525 wave numbers, respectively. The la-
recruited prior to treatment. Abnormal sites ser power levels are within the limits given by
and contralateral normal sites were measured. the ANSI standard for ocular exposure (2.7 rotenoid molecules. It is clear that a rapid ob-
AU measurement sites from patients were bi- J/cm2), and the obtained signals are free of jective technique to noninvasively quantify
opsied for pathologic analysis. In addition, potentially interfering signals from other mol- macular pigment levels in human subjects
normal volunteers with no history and no ob- ecules in the retinal tissue. would be a major advance in the study and
served oral lesions were evaluated. Thirteen Of the approximately 10 carotenoid pig- possiible prevention of macular degeneration
patients and three normal subjects have been ments found in normal human serum, the spe- diseases.
measured. cies lutein and zeaxanthin are concentrated in A typical Raman carotenoid spectrum is
Figure 2 shows two typical EEM contour high amounts in the cells of the human shown in Fig. 1 for a flat-mounted human
plots from a normal and an abnormal area of macula, which is a -5 mm diameter area of the retina. We observe two strong Stokes-shifted
retina in which the visual acuity is highest. Raman lines due to carbon-carbon single-
the dorsal tongue. Consistent with previous
These carotenoids give the macula a character- bond and double-bond stretch vibrations of
findings: fluorescence from abnormal sites is
istic yellow coloration, and it is speculated that the carotenoids at 1158 and 1528 cm-', re-
less intense compared with normal sites. Addi-
-
tionally fluorescence at XeYCltatlOn 380 nm
their absorption may function as filter to at-
tenuate photochemical damage and/or image
spectively, and a weaker signal at 1008 cm-'
from methyl components. All Raman signals
shows a wider FWHM at the normal site. Ab- degradation under bright UV/blue light expo- are .superimposed on a weak fluorescence
sorption bands of oxygenated hemoglobin sures. In addition, they are thought to act as background. The peaks at -1159 and -1524
(420, 540, 580 nm) and deoxygenated hemo- free-radical scavenging antioxidants. Studies cm-' are obtained with good signal-to-noise
globin (420, 560 nm) can be observed. The have shown that there may be a link between ratio when the beam is aimed at the foveal and
corresponding reflectance data (Fig. 3) are macular degenerative diseases, the leading parafoveal areas (traces a and b), and they de-
relatively featureless above 600 nm. The cause of blindness in the elderly in the United crease by at least a factor of 30 as the beam is
double absorption bands of oxy-hemoglobin States, and the presence or absence of the ca- moved toward the peripheral retina (trace c).
at 540 and 580 nm are present in the spectrum
of normal tissue, while that of abnormal tissue
shows a deoxyhemoglobin absorption band at
560 nm. The reflectance of the abnormal site at
480 nm is greater than that of the normal site.
We have demonstrated the acquisition of
EEMs in combination with spatially resolved
reflectance measurements in the oral cavity.
Findings in the reflectance and fluorescence
spectra can be correlated to pathology, and 400
used to determine the optimal combination of
techniques and wavelengths for detection of
early stage oral cavity neoplasia.
*Department of Head and Neck Surgery, The
University of Texas M.D. Anderson Cancer Cen-
ter, Houston, Texas
**Department of Pathology, The University of 200
Texas M.D. Anderson Cancer Center, Houston,
Texas
1. Ramanujam et al., Photochem. Photobiol.
64(4), 720 (1996).
2. Mourant et al., Lasers Surgery Medicine,
XI0 A
17(4), 350 (1995). 0
3. Gillenwater, Noninvasive diagnosis of oral
loo0 1200 1400 1600
neoplasia based onfluorescence spectroscopy
and native tissue autofluorescence, pre-
sented at International conference on Rarmn / arrl
Head and Neck Cancer, Toronto, Canada, CW03 Fig. 1. Resonance Raman spectra of flat-mounted human retina, obtained under excitation
August 1996. with 4880 8, light in the center (trace a), 0.5 mm away from the center (trace b) and the periphery (trace
4. Dhingra et al., Arch Otolaryngol Head c) of the macula. The intensity scale of trace (c) is expanded by a factor of 10 for clarity. Illumination
Neck Surg. 122,1181-1186 (1996). conditions: 4880 8,,40 mW, 300 pm spot size, 9 s.
308 / CLEO’98 / WEDNESDAY AFTERNOON
This behavior correlates well with the known
distribution of carotenoids in the human
retina as determined by high-performance liq-
uid chromatography (HPLC) or psychophysi-
cally. We find that other human ocular struc-
tures such as the cornea, lens, vitreous, retinal
pigment epithelium, choroid, and sclera do
not generate any detectable or interfering Ra-
man signals under comparable conditions.
We were also able to demonstrate a linear
correlation between Raman signal strength
and actual macular carotenoid levels as deter-
mined by HPLC and shown in Fig. 2. The
current development of an apparatus suitable
for measurements on living eyes will be dis-
cussed.
*Moran Eye Center, University of Utah, Salt
Lake City, Utah 84123
CW04 5 3 0 pm
Wholefield fluorescence lifetime
imaging with picosecond resolution for
biomedicine
K. Dowling, M.J. Dayel, P.M.W. French,
P. Vourdas,* M.J. Lever,*
A.K.L. Dymoke-Bradshaw,**J.D. Hares,**
Femtosecond Optics Group, Physics
Department, Imperial College, London SW7
2BZ, United Kingdom; E-mail: paul.
french@ic.ac.uk
Fluorescence lifetime measurements permit
both detection of specific fluorophores and
monitoring of their local environment for bio-
medical and other applications including mi-
croscopy, biotechnology, and process moni-
toring. Fluorescence lifetime imaging (FLIM)
is particularly exciting because it can provide
noninvasive functional/diagnostic imaging by
exploiting the sensitivity of fluorescence life-
time to the local environment (e.g. [Ca”], pH
etc.).”’ The recent development of user-
friendly and relatively portable ultrafast laser
technology and the availability of ultrafast
gated optical image intensifiers (GOIs) mean
that this technology can be readily applied to
almost any optical imaging modality including
microscopy and endoscopy. We report a FLIM
system,’ based on a GO1 (Kentech Gated Op-
tical Imager) with a temporal response of 90
ps, which uses pulses of -1 pJ energy. This is
compatible with our all-solid-state diode-
pumped Cr:LiSAF oscillator-amplifier sys-
tem: potentially providing a portable and
relatively inexpensive instrument.
Using the apparatus shown in Fig. 1, fluo-
-
rescent samples were excited by 10 ps pulses
at415nm.AFLIMmapisproducedbyrecord-
ing images of the fluorescence at different de-
lays after excitation, and performing a least-
squares fit to each point in the field ofview. For
simple fluorophore distributions we have
demonstrated near-real-time FLIM with an
update time of <3 s. In order to investigate the
minimum detectable fluorescence lifetime dif-
ference, we prepared a phantom consisting of
four pipettes containing the laser dye DASPI in
different ethanol/glycerol solutions. Changing
the viscosity of the solvent changes the fluores-
cence lifetime of the dye.5 Figure 2 shows the
resulting FLIM map, and a transverse cross
section, displaying lifetime differences as short
..................... .............-
,.. ........-.
1 415 nm, - I O ps
L. .........
._.__.
CW04 Fig. 1. Experimentalsetup.
.._,
position I arb. units
(b)
CW04 Fig. 2. (a) FLIM map of DASPI in dif-
ferent viscosity solvents. Solvents, from left to
right: (50:50)-(54:46)-(50:50)-(54:46):(g1yc-
ero1:ethanol) and (b) a horizontal cross section
through the center of the FLIM map.
-
as 10ps. We note that the maximum lifetime
that may be imaged using this apparatus is
limited only by the repetition rate of the laser
system. Thus this FLIM system exhibits excel-
lent temporal resolution and dynamic range.
For pipette experiments, the two spatial di-
mensions of the detector mean that it is pos-
sible to compare multiple samples and to re-
solve spectrally as well as temporally in a single
acquisition. This functionality has been dem-
onstrated.
We have begun applying this FLIM system
to biomedical imaging and have investigated
the autofluorescence of different tissue con-
stituents. We have observed that, for excitation
at 415 nm, the fluorescence of elastin and col-
lagen exhibits a double exponential decay,
which we believe to arise from the cross-
linkage of the fibers. Figure 3 shows (a) a time-
gated image and (b, c) the FLIM maps corre-
sponding to the first and second components
(C)
CW04 Fig. 3. FLIM of tissue. Samples, from
left to right, Elastin-Aorta-Elastin-
Collagen-Aorta. (a)Time gated image at peak of
fluorescence decay, (b) FLIM map showing the
short-lived component of the double-
exponential fluorescence decay, and (c) the long-
lived component.
ofthe fluorescence decays of samples of elastin,
collagen and aorta. It will be seen that the
FLIM maps provide a means of distinguishing
between these different tissue types, (even
though their spectral fluorescence profiles
showed no significant difference for this exci-
tation wavelength). These early results indicate
the potential of FLIM as a diagnostic tool to
detect and monitor specific tissue types or con-
ditions.
*Departmentof Biological and Medical Systems,
Imperial College, London SW7 2A2, United
Kingdom
**Kentech Instruments Ltd., Unit 9, Hall Farm
Workshops, South Moreton, Didcot, Oxon.,
OX1 1 9AG, United Kingdom
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Minami, Appl. Spectrosc. 45,360 (1991).
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son, Methods in Enzymology 240, 723
(1994).
3. K. Dowling, S.C.W. Hyde, J.C. Dainty,
P.M.W. French, Opt. Commun. 135, 27
(1997).
4. S.C.W. Hyde, N.P. Barry, R. Mellish,
P.M.W. French, J.R. Taylor, C.J. van der
Poel, A. Valster, Opt. Lett. 20, 160 (1995).
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(1983).