The specificity of molecular and functional photoacoustic (PA) images depends on the accuracy of the photoacoustic absorption spectroscopy. The PA signal is proportional to the product of the optical absorption coefficient and local light fluence; quantitative PA measurements of the optical absorption coefficient therefore require an accurate estimation of optical fluence. Light-modeling aided by diffuse optical tomography (DOT) can be used to map the required fluence and to reduce errors in traditional PA spectroscopic analysis. As a proof-of-concept, we designed a tissue-mimicking phantom to demonstrate how fluence-related artifacts in PA images can lead to misrepresentations of tissue properties. To correct for these inaccuracies, the internal fluence in the tissue phantom was estimated by using DOT to reconstruct spatial distributions of the absorption and reduced scattering coefficients of multiple targets within the phantom. The derived fluence map, which only consisted of low spatial frequency components, was used to correct PA images of the phantom. Once calibrated to a known absorber, this method reduced errors in estimated absorption coefficients from 33% to 6%. These results experimentally demonstrate that combining DOT with PA imaging can significantly reduce fluence-related errors in PA images, while producing quantitatively accurate, high-resolution images of the optical absorption coefficient.
The specificity of both molecular and functional
photoacoustic (PA) images depends on the accuracy of the
photoacoustic absorption spectroscopy. Because the PA signal is
a product of both the optical absorption coefficient and the local
light fluence, quantitative PA measurements of absorption
require an accurate estimate of the optical fluence. Lightmodeling
aided by diffuse optical tomography (DOT) methods
can be used to provide the required fluence map and to reduce
errors in traditional PA spectroscopic analysis. As a proof-ofconcept,
we designed a phantom to demonstrate artifacts
commonly found in photoacoustic tomography (PAT) and how
fluence-related artifacts in PAT images can lead to
misrepresentations of tissue properties. Specifically, we show that
without accounting for fluence-related inhomogeneities in our
phantom, errors in estimates of the absorption coefficient from a
PAT image were as much as 33%. To correct for this problem,
DOT was used to reconstruct spatial distributions of the
absorption coefficients of the phantom, and along with the
surface fluence distribution from the PAT system, we calculated
the fluence everywhere in the phantom. This fluence map was
used to correct PAT images of the phantom, reducing the error in
the estimated absorption coefficient from the PAT image to less
than 5%. Thus, we demonstrate experimentally that combining
DOT with PAT can significantly reduce fluence-related errors in
PAT images, as well as produce quantitatively accurate, highresolution
images of the optical absorption coefficient.
Chlorin-bacteriochlorin dyads as a new class of near-infrared fluorophores were synthesized and
spectroscopically characterized. Each dyad is comprised of a chlorin macrocycle (free base or zinc chelate) as an energy
donor (and absorber) and a free base bacteriochlorin as an energy acceptor (and emitter). Excitation of the chlorin (λ=
650 nm, zinc chelate; 675 nm, free base) results in fast (5 ps) and nearly quantitative (>99%) energy transfer to the
adjacent bacteriochlorin moiety, and consequently bacteriochlorin fluorescence (λ= 760 nm). Thus, each chlorinbacteriochlorin
dyad behaves as a single chromophore, with a large effective Stokes shift (85 or 110 nm), a significant
fluorescence quantum yield (Φf = 0.19), long excited-state lifetime (τ = 5.4 ns), narrow excitation and emission bands
(<20 nm), and high chemical stability.
Imaging experiments performed using phantoms show that the chlorin-bacteriochlorin dyads exhibit a range of
superior properties compare with commercially available imaging dyes. While the latter are six-fold brighter (comparing ε•Φf values), the chlorin-bacteriochlorin dyads exhibit narrower excitation and emission bands and larger Stokes shift,
therefore allowing more efficient and selective excitation and detection of fluorescence. The high selectivity is further
demonstrated with in vivo imaging studies using mice. This selectivity together with the tunability of absorption and
emission wavelengths using substituent effects under synthetic control make the chlorin-bacteriochlorin dyads ideal
candidates for multicolor imaging applications. In addition, the long fluorescence lifetimes make those probes suitable
for lifetime-imaging applications.
Local molecular and physiological processes can be imaged in vivo through perturbations in the fluorescence lifetime (FLT) of optical imaging agents. In addition to providing functional information, FLT methods can quantify specific molecular events and multiplex diagnostic and prognostic information. We have developed a fluorescence lifetime diffuse optical tomography (DOT) system for in vivo preclinical imaging. Data is captured using a time-resolved intensified charge coupled device (ICCD) system to measure fluorescence excitation and emission in the time domain. Data is then converted to the frequency domain, and we simultaneously reconstruct images of yield and lifetime using an extension to the normalized Born approach. By using differential phase measurements, we demonstrate DOT imaging of short lifetimes (from 350 ps) with high precision (±5 ps). Furthermore, this system retains the efficiency, speed, and flexibility of transmission geometry DOT. We demonstrate feasibility of FLT-DOT through a progressive series of experiments. Lifetime range and repeatability are first measured in phantoms. Imaging of subcutaneous implants then verifies the FLT-DOT approach in vivo in the presence of inhomogeneous optical properties. Use in a common research scenario is ultimately demonstrated by imaging accumulation of a targeted near-infrared (NIR) fluorescent-labeled peptide probe (cypate-RGD) in a mouse with a subcutaneous tumor.
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