High-resolution images in deep tissue are obtained using photoacoustic images of Förster resonance energy transfer, overcoming the problem of light-scattering effects in some imaging techniques

Wang, Lihong

Wang, Yu

Caltech Authors - Main

10.1117/2.1201211.004595
Photoacoustic microscopy using
Fo¨rster resonance energy
transfer
Yu Wang and Lihong Wang
High-resolution images in deep tissue are obtained using photoacoustic
images of Fo¨rster resonance energy transfer, overcoming the problem of
light-scattering effects in some imaging techniques.
Fo¨rster resonance energy transfer (FRET) is a physical process in
which energy is transferred from an excited donor fluorophore
to adjacent chromophores non-radiatively.1 Because the trans-
fer rate is sensitive to the distance between donor and acceptor,
FRET provides amolecular ’ruler’ for measuring the distance be-
tween biomolecules. The process is valuable to the understand-
ing of protein interactions and conformational changes, and we
can use it, in vivo, to explore protease activity, protein misfold-
ing, and intracellular calcium.
FRET manifests through the reduction of donor fluorescence
emission as a result of non-radiative energy transfer to the ac-
ceptor, so fluorescence imaging is well suited to produce FRET
images. However, the intensive light scattering that occurs be-
yond the depth of a few hundred microns precludes fluores-
cence imaging of FRET at greater depths and with high spatial
resolution.
We have developed high-resolution photoacousticmicroscopy
of FRET in deep tissue.2 Photoacoustic imaging is based on
detecting non-radiative decay processes ultrasonically. When a
fluorophore is excited, it undergoes either transitions of flu-
orescence emission, or it experiences fast non-radiative de-
cay to the ground state. FRET provides a third mechanism
for excited state decay, where energy is transferred from
a donor fluorophore to an acceptor chromophore by non-
radiative dipole-dipole coupling. If a non-fluorescent accep-
tor is chosen, all the transferred energy must decay through
the acceptor’s non-radiative pathway. The non-radiative de-
cay converts the excited molecular state energy into heat
and subsequent thermoelastic expansion, generating acoustic
waves (photoacoustic signals) in the medium. Therefore, when
Figure 1. Schematic diagram of a dual-modality fluorescence and pho-
toacoustic microscope used to demonstrate acoustic imaging of Fo¨rster
resonance energy transfer (FRET). PMT: Photomultiplier.
FRET occurs, an increase of photoacoustic signal strength
accompanies the donor fluorescence quenching. While flu-
orescence imaging observes a decrease in donor fluores-
cence or an increase in acceptor fluorescence, photoacoustic
imaging visualizes the increase in pressure generated by the ac-
ceptor. Photoacoustic imaging enables deep penetration because
the magnitude of acoustic scattering in tissue is three times less
than that of optical scattering. Moreover, photoacoustic imaging
is scalable with optical illumination and ultrasonic detection. It
can provide submicron resolution at a depth of 1cm while main-
taining a high depth-to-resolution ratio.3
We demonstrated photoacoustic imaging of FRET in solutions
using a dual-modality photoacoustic and fluorescence confocal
microscope (see Figure 1).4 We prepared seven stock ethanol
solutions with concentrations of fluorescent donor Rhodamine
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10.1117/2.1201211.004595 Page 2/3
6G (R6G) and non-fluorescent acceptor 1,3 ’diethyl-4,2’ quinoly-
oxacarbocyanide iodide (DQOCI), as in Figure 2(a). Figure 2(b)
and (c) shows the fluorescence and photoacoustic images ac-
quired at 523nm. When the donor R6G was excited, we detected
fluorescence and photoacoustic emissions in the presence of
different concentrations of acceptor DQOCI. Compared with a
solution containing only donor R6G (tube 1), the mixtures con-
taining both donor R6G and acceptor DQOCI had diminished
fluorescence signals. Increasing the quantity of acceptor DQOCI
made fluorescence quenching more effective, and it led to a
higher photoacoustic signal (tubes 3, 5 and 7). The FRET effi-
ciency mappings based on the fluorescence and photoacoustic
measurements are shown in Figure 2(d) and (e), respectively. The
color-coded images display the increase in FRET efficiency due
to the increase in the acceptor concentration.
Figure 2. (a) Photograph of the tube phantom and table showing con-
centrations of donor Rhodamine 6G (R6G) and acceptor DQOCI. (b)
Fluorescence microscopic image acquired at 523nm. (c) Photoacoustic
microscopic image acquired at 523nm. (d) FRET efficiency maps
acquired using the fluorescence microscope. (e) FRET efficiency maps
acquired using the photoacoustic microscope.
Figure 3. (a) Fluorescence microscopic image, acquired at 523nm, of the
tube phantom with overlaid mouse skin tissue. (b) Cross-sectional B-
scan photoacoustic microscopic image of the tubes acquired at 523nm.
(c) Photoacoustic microscopic image acquired at 523nm. (d) FRET ef-
ficiency map acquired using the photoacoustic microscope.
To compare the photoacoustic and fluorescence approaches
for deep tissue FRET imaging, we overlaid intervening mouse
skin tissue on the samples. The tubes detected in the fluores-
cence confocal image were severely blurred by light scattering
in the tissue: see Figure 3(a). The photoacoustic cross sectional
B-scan image of the tubes shows that the overlaid mouse skin
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10.1117/2.1201211.004595 Page 3/3
had a thickness of 1mm: see Figure 3(b). Again, the pho-
toacoustic images of Figure 3(c) show FRET quenching of the
donor R6G fluorescence by acceptor DQOCI. Photoacoustic mi-
croscopy achieves FRET imaging of the tissue phantom, as in
Figure 3(d), whereas we cannot generate a similar map from flu-
orescence microscopy because of its poor signal-to-noise ratio:
see Figure 3(a).
Photoacoustic microscopy has been used to image FRET ef-
ficiencies through a 1mm skin tissue, and further study has
demonstrated a penetration of 10mm in scattering tissue. Based
on the relative increase of photoacoustic signals, we can quantify
absolute FRET efficiency. Compared with confocal microscopy,
photoacoustic microscopy offers better penetration into scatter-
ing biological tissue. In our future work, we will extend photoa-
coustic FRET imaging to in vivo animal studies.
This work was sponsored in part by National Institute of Health grants
R01 EB000712, R01 EB008085, R01 CA134539, U54 CA136398, R01
CA157277, and R01 CA159959. Lihong Wang has a financial interest
in Microphotoacoustics Inc. and Endra Inc., although these companies
did not support this work.
Author Information
Yu Wang and Lihong Wang
Washington University in St. Louis
St. Louis, MO
Yu Wang received his BS in physics from the University of
Science and Technology of China in 2008. He is pursuing a PhD
in the Department of Biomedical Engineering under the super-
vision of Professor Lihong Wang.
Lihong Wang holds the Gene K. Beare Distinguished Profes-
sorship of Biomedical Engineering. His laboratory invented
3D photoacoustic microscopy, enabling non-invasive biochem-
ical, functional, and molecular imaging in living tissue at high
resolution.
References
1. A. Periasamy, S. S. Vogel, and R. M. Clegg, FRET 65: a celebration of Forster, J.
Biomed. Opt. 17 (1), p. 011001, 2012. doi:10.1117/1.JBO.17.1.011001
2. Y. Wang and L. V. Wang, Fo¨rster resonance energy transfer photoacoustic microscopy,
J. Biomed. Opt. 17 (8), p. 086007, 2012. doi:10.1117/1.JBO.17.8.086007
3. L. V. Wang and S. Hu, Photoacoustic tomography: in vivo imaging from organelles to
organs, Science 335 (6075), pp. 1458–1462, 2012. doi:10.1126/science.1216210
4. Y. Wang, K. Maslov, C. Kim, S. Hu, and L. V. Wang, Integrated photoacoustic and
fluorescence confocal microscopy, IEEE Trans. Biomed. Eng. 57 (10), pp. 2576–2578,
2010. doi:10.1109/TBME.2010.2059026
c 2012 SPIE


Photoacoustic microscopy using Förster resonance energy transfer

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Photoacoustic microscopy using Förster resonance energy transfer

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