Simultaneously achieving high signal-to-noise ratio (SNR) (or sensitivity) and high resolution is desired in biomedical imaging. However, conventional imaging modality has a tradeoff between SNR (or sensitivity) and resolution. We developed a method to simultaneously achieve high SNR (or sensitivity) and high resolution for fluorescence imaging in deep tissue. We first introduce a recently developed deep-tissue high-resolution imaging technique termed as ultrasound-switchable fluorescence (USF). An approach of modulating ultrasound exposure time is adopted to increase the detectability of the USF signal. The control parameters of modulation of ultrasound—such as (1) frequency, (2) duty cycle, and (3) exposure duration—are varied to study their influence on the USF signal and SNR. We conclude that high SNR can be achieved by modulating ultrasound exposure without sacrificing the spatial resolution. This is important for future fluorescence molecular imaging of cancer in deep tissue.
A fluorescence resonance energy transfer (FRET)-based microbubble contrast agent system was designed to experimentally demonstrate the concept of ultrasound-modulated fluorescence (UMF). Microbubbles were simultaneously labeled with donor and acceptor fluorophores on the surface to minimize self-quenching and maximize FRET. In response to ultrasound, the quenching efficiency was greatly modulated by changing the distance between the donor and acceptor molecules through microbubble size oscillations. Both donors and acceptors exhibited UMF on individual microbubbles. The UMF strength of the donor was more significant compared to that of the acceptor. Furthermore, the UMF of the donor was observed from a microbubble solution in a turbid media. This study exploits the feasibility of donor–acceptor labeled microbubbles as UMF contrast agents.
Bioaffinity conjugation between streptavidin (SA) and biotin has been widely used to link donors and acceptors for investigating the distance-dependent Förster resonance energy transfer (FRET). When studying a commonly used FRET system of (QD-SA)-(biotin-DNA-dye) [donor: quantum dot (QD); acceptor: small organic fluorescent dye; and linker: deoxyribose nucleic acid (DNA) molecule via SA-biotin conjugation], however, a contradictory finding was recently reported in the literature. It was found that the FRET lost its dependence on the number of DNA base pairs when using a phosphate-buffered saline (PBS) solution. We found that the conflicted results were caused by the ionic strength of the adopted buffer solutions. Our results suggest that the dependent FRET on the number of DNA bases is favorable in a low-ionic-strength buffer, whereas in relatively high-ionic-strength buffers, the FRET loses the DNA length dependence. We propose that the independence is mainly caused by the conformational change of DNA molecules from a stretched to a coiled mode when the cations in the high-ionic-strength buffer neutralize the negatively charged backbone of DNA molecules, thereby bringing the acceptors close to the donors.
Ultrasound-modulated fluorescence (UMF) imaging has been proposed to provide fluorescent contrast while maintaining ultrasound resolution in an optical-scattering medium (such as biological tissue). The major challenge is to extract the weakly modulated fluorescent signal from a bright and unmodulated background. UMF was experimentally demonstrated based on fluorophore-labeled microbubble contrast agents. These contrast agents were produced by conjugating N-hydroxysuccinimide (NHS)-ester-attached fluorophores on the surface of amine-functionalized microbubbles. The fluorophore surface concentration was controlled so that a significant self-quenching effect occurred when no ultrasound was applied. The intensity of the fluorescent emission was modulated when microbubbles were oscillated by ultrasound pulses, presented as UMF signal. Our results demonstrated that the UMF signals were highly dependent on the microbubbles’ oscillation amplitude and the initial surface fluorophore-quenching status. A maximum of ∼42% UMF modulation depth was achieved with a single microbubble under an ultrasound peak-to-peak pressure of 675 kPa. Further, UMF was detected from a 500-μm tube filled with contrast agents in water and scattering media with ultrasound resolution. These results indicate that ultrasound-modulated fluorescent microbubble contrast agents can potentially be used for fluorescence-based molecular imaging with ultrasound resolution in the future.
The question of whether particle size affects modulation efficiency, defined as the ratio of ultrasound-modulated
fluorescence (UMF) signal to DC (direct current) signal, of the fluorescence emission from four different sized
fluorescent particles was investigated experimentally. The four particles are streptavidin-conjugated Alexa Fluo 647 (~5
nm in diameter) and three carboxylate-modified fluorescent microspheres (FM) with different diameters of 0.02, 0.2, and
1.0 μm. Modulation efficiency was evaluated as a function of the fluorophore size and fluorophore concentration. The
modulation efficiency was improved about two times when the size of the fluorescent particles is increased from 5 nm to
1 μm. This result implies that using large fluorescence particles can slightly improve the modulation efficiency but the
improvement is limited.
We report experimentally observed ultrasound-modulated fluorescence (UMF) from a submillimeter tube filled with rhodamine B aqueous solution. The tube was submerged in water and a scattering medium. Based on the measured data, we find that the UMF signals might be generated from three mechanisms: modulation of the excitation light, modulation of the emission light, and modulation of the properties of fluorophore. In addition, a linear relationship between the UMF and the drive voltage applied to the ultrasound transducer is found.
We demonstrate the feasibility of fluorescence imaging of deeply seated tumors using mice injected with an angiogenesis tracer, a vascular endothelial growth factor conjugated with the infrared dye cyanine 7 (VEGF/Cy7). Our optical-only imaging reconstruction method separately estimates the target depth, and then applies this information to reconstruct functional information such as fluorophore concentration. Fluorescence targets with concentrations as low as sub-25 nM are well reconstructed at depths up to 2 cm in both homogeneous and heterogeneous media with this technique.
Ultrasound-modulated fluorescence from a fluorophore-quencher-labeled microbubble system driven by a single ultrasound pulse was theoretically quantified by solving a modified Herring equation (for bubble oscillation), a two-energy-level rate equation (for fluorophore excitation), and a diffusion equation (for light propagation in tissue). The efficiency of quenching caused by fluorescence resonance energy transfer (FRET) between the fluorophore and the quencher was modulated when the microbubble oscillates in size driven by the ultrasound pulse. Both intensity- and lifetime-based imaging methods are discussed in three different illumination modes of the excitation light: continuous wave (DC), frequency domain (FD), and time domain (TD). Results show that microbubble expansion opens a time period during which the quenching efficiency is dramatically reduced so that the emitted fluorescence strength and fluorophore lifetime are significantly increased. The modulation efficiency may even reach 100%. In addition, an important finding in this study is that in TD illumination mode, the modulated fluorescence photons may be temporally separated from the unmodulated photons, which makes the modulation efficiency limited only by thermal noise of the measurement system.
Ultrasound-modulated fluorescence from a fluorophore-quencher labeled microbubble system driven
by a single ultrasound pulse was theoretically quantified by solving a modified Herring equation (for
bubble oscillation), a two-energy-level rate equation (for fluorophore excitation), and a diffusion
equation (for light propagation in tissue). The efficiency of quenching caused by fluorescence
resonance energy transfer (FRET) between the fluorophore and the quencher was modulated when
the microbubble oscillates in size driven by the ultrasound pulse. Both intensity- and lifetime-based
imaging methods are discussed. An important finding in this study is that ultrasound-modulated
fluorescent photons may be temporally separated from the
un-modulated fluorescent photons if a
super-short laser pulse is adopted. This result implies that the modulation efficiency may only be
limited by thermal noise of the measurement system.
Radiative transport in the delta-P1 and delta-P3 approximations with an extrapolated boundary condition
combined with the reciprocity was demonstrated providing accurate estimations for optical perturbations by comparing
with experimental data in a small volume of a liquid tissue phantom. For an absorbing target submerged in a
homogeneous medium with an albedo of 0.75, both approximations estimated the optical perturbation with acceptable
accuracy when the depth of the target is larger than 1.5 mm. For a fluorescent target, the two approximations estimated
the perturbations with high accuracy for both shallow and deep regions. Therefore, these two approximations combined
with the reciprocity were proposed for rapid tomographic image reconstruction in a small volume or superficial tissues.
We present a robust technique for diffuse optical fluorescence imaging of tumors in mice and tissue
simulating fluorescence phantoms. The detection optics, which is a crucial part of a frequency domain
fluorescence imaging system, with appropriate optical filters for efficient rejection of the excitation light, is
demonstrated. The image reconstruction is divided into two parts; i.e. reconstructing the target locations
such as size and position, and reconstructing the functional information such as fluorophore concentration
and image reconstruction. The structural parameters i.e. tumor size and locations of the targets are
recovered by a chi-square fitting technique by fitting the experimental data into analytically generated data.
Having the structural information beforehand, the images are reconstructed by using our dual-mesh
technique. The fluorescence images of targets of few tens of nanomolar fluorophore concentrations in both
homogeneous and heterogeneous media are reconstructed in this study.
Shallow lesions less than 1.5-cm deep are frequently seen in breast patients when they are scanned in reflection geometry. Two boundary conditions are compared for imaging shallow lesions, and a new probe design is introduced. A partial reflection boundary condition is suitable for imaging shallow lesions less than 1.0-cm deep; whereas an absorption boundary condition is desirable for imaging lesions more than 1.5-cm deep. Our new probe design incorporates either a partial reflection boundary or an absorption boundary based on a priori knowledge of lesion depth provided by coregistered real-time ultrasound images. An angled source is introduced to further improve the illumination of the region between 1.0- to 1.5-cm depths. Simulation, phantom, and freshly excised mouse tumor experiments demonstrate that targets located at different depths can be uniformly reconstructed. A clinical example is given to demonstrate the utility of this new approach for optimally probing lesions located at different depths.
We propose a self-normalized scanning fluorescence diffuse optical tomography technique. The method requires a single
modulated light source for excitation and multiple detector pairs symmetrically located at positions beside the source.
The amplitude ratio and phase difference between the two detectors of each pair are measured as the source and detectors
are scanned through the region of interest. It has been observed that the phase difference profile along a scanning line
depends on target depth rather than fluorophore concentration enabling estimation of the target depth from the phase
difference before reconstruction of the fluorophore concentration. The depth of a cylindrical target with a target-to-background
contrast of 0.2:0.01 (Cy5.5) was varied from 0.7cm to 1.8cm, and the estimated depths were very close to
their expected values with 15% maximum error. Based on the estimated target depth, the imaging volume can be
segmented into a smaller region surrounding the target and a larger background region enabling the use of a dual-zone mesh
based image reconstruction. Fluorophore concentration was reconstructed by using both amplitude ratio and phase
difference between the detector pairs. The reconstructed fluorophore concentration has achieved 90% accuracy.
Because the amplitude ratio is dimensionless and the phase difference is a relative value, this technique is self-normalized
and sensitive to low-contrast heterogeneity of fluorophore concentration in a turbid medium. In addition,
based on the estimated target position and depth, the dual-zone mesh reconstruction scheme has significantly improved
the reconstruction accuracy of fluorophore concentration.
In this paper, we propose a partially reflecting boundary method for diffusive optical imaging of shallow targets in turbid media. An appropriate range of the effective reflection coefficient Reff has been suggested between 0.0 and 0.7, in which the extrapolated boundary condition can be used to simplify the forward model. For a shallow target of approximately 0.5 cm deep from the surface, experimental data acquired with a reflecting boundary of Reff ≈ 0.6 lead to significant improvement in the image quality compared with that from an absorbing boundary of Reff ≈ 0.
Emission and absorption properties of indocyanine green (ICG) in Intralipid solution have been investigated. The study is focused on relatively low ICG concentration at a range of 0 to 20 µM. A diffusion model was used to analyze the emission properties of ICG solution at different concentrations. In the low-concentration region, the emission strength increases with the concentration of ICG, while in the high-concentration region, the emission decreases with the concentration. In general, a maximum of emission strength exists and its position (concentration) depends on the wavelength of the excitation light, the distance between the source and the detector, and the sample geometry and size. A so-called "inner-cell-effect" and re-absorption of emission photons are found to contribute to the decay of emission strength. Also, in the concentration range of 0 to 2 µM, ICG solution always has a higher absorption coefficient at wavelength 830 nm than that at 660 nm, which is quite different from the ICG in water case.
In this paper, a 3D dual-mesh imaging reconstruction method is demonstrated, which can reconstruct absorption and scattering coefficients simultaneously. In the dual-mesh scheme, the total number of voxels with unknown absorption and scattering perturbations are maintained on the same order of total measurements by using a fine grid for target region and a coarse grid for background region. Certain row/column normalization has been applied to alleviate the crosstalk between the absorption coefficient and scattering coefficient, and to minimize the depth dependent problem. Experimental results of targets with different absorption and scattering contrasts have shown that accurate reconstruction of both absorption and scattering coefficients can be achieved.
In this paper, we have applied a perturbative expanding method to the hopping model, and studied the coupling effects of modulation depth between two pieces of photorefractive phase gratings stored in one point with an external applied dc field. The coupling equations of modulation depth and their steady solution have be derived. It has been found that the spatial -charge-filed of one of the two gratings is seriously affected by the modulation depth of other gratings.
Chalcogenide alloys SbxSey are being applied to phase-change, reversible optical storage. The non- stoichiometric compound are researched displaying their interesting phenomena before and after anneal in a furnace, which were characterized by the X-ray diffraction technology. The composition of the Se richer than Sb will result the film in amorphous state in the room temperature evaporation. The Sb2Se3 crystallized out from the non-stoichiometric alloys have the lower crystallization temperature compared with the stoichiometric alloys Sb2Se3. Double layers consisting of Sb film deposited on Se film was investigated which revealed its special characteristics. The sublimation and the diffusion of the atoms during annealing in the lower temperature have the important effects on the material's crystallization behavior, the great kinetic energy of the atoms will result in the crystallization of Se and Sb2Se3 and, the lower crystallization temperature is just what we expected in the laser recording and for the optical-data storage. An important conclusion can be made from the experiments, the crystallization can be finished by adjacent atomic diffusion. The reason for optical crystallization is also discussed.
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