Baohong Yuan

A system for high-resolution depth-resolved optical imaging of fluorescence and absorption contrast

Laminar optical tomography (LOT) is a new three-dimensional in vivo functional optical imaging technique. Adopting a microscopy-based setup and diffuse optical tomography (DOT) imaging principles, LOT can perform both absorption- and fluorescence-contrast imaging with higher resolution (100-200 microm) than DOT and deeper penetration (2-3 mm) than laser scanning microscopy. These features, as well as a large field of view and acquisition speeds up to 100 frames per second, make LOT suitable for depth-resolved imaging of stratified tissues such as retina, skin, endothelial tissues and the cortex of the brain. In this paper, we provide a detailed description of a new LOT system design capable of imaging both absorption and fluorescence contrast, and present characterization of its performance using phantom studies.

Optimal probing of optical contrast of breast lesions of different size located at different depths by US localization

We report a frequency domain optical tomography system utilizing three RF modulation frequencies, which are optimized for probing breast lesions of different size located at different depths. A real-time co-registered ultrasound scanner is used to provide on-site estimation of lesion size and location. Based on the lesion information, an optimal light modulation frequency can be selected, which may yield more accurate estimates of lesion angiogenesis and hypoxia. Phantom experiments have demonstrated that a high modulation frequency, such as 350Mhz, is preferable for probing small lesions closer to the surface while a low modulation frequency, such as 50Mhz, is desirable for imaging deeper and larger lesions. A clinical example of a large invasive carcinoma is presented to demonstrate the application of this novel technique.

Ultrasound-modulated fluorescence based on a fluorophore-quencher-labeled microbubble 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 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.

Microbubble-enhanced ultrasound-modulated fluorescence in a turbid medium

The feasibility of using ultrasound to modulate fluorescence in a turbid medium is still in debate due to the difficulty of detecting the modulated signal. We have demonstrated a system that could detect the weak signals of ultrasound-modulated fluorescence (UMF) by using a broadband lock-in amplifier and microbubbles as enhancement agents. By detecting the microbubble-enhanced UMF signal, a sub-millimeter fluorescent tube submerged in a turbid medium with a depth of 2 cm has been clearly observed with an ultrasonic spatial resolution. The modulation efficiency was significantly improved by using microbubbles, and was found to linearly increase with the drive voltage applied to the ultrasound transducer and the fluorophore concentration within the range adopted in this study. Possible modulation mechanisms are discussed.

Mechanisms of the ultrasonic modulation of fluorescence in turbid media

To understand the modulation mechanisms of fluorescence emission induced by ultrasonic waves in turbid media, a mathematical model is proposed and compared with the recent experimental observations of Kobayashi et al. [Appl. Phys. Lett. 89, 181102 (2006)]. Modulation of fluorophore concentration is considered as the source of the oscillation of fluorescence signals when fluorophore concentration is low enough so that quenching effects can be ignored. By solving the rate equation and photon diffusion equation, quantitative solutions are given to quantify the modulation strength. Our calculations predict that the modulation depth (the ratio of the modulated signal strength to the unmodulated signal strength) can reach 10(-4) when ultrasonic pressure with the order of magnitude of megapascals is applied in the ultrasound focal zone. Our model explains the relationship between the modulation strength and the average fluorophore concentration and also predicts a method to measure or image fluorescence lifetime in the turbid medium. When fluorophore concentration is high enough so that fluorescence quenching occurs, the fluorescence modulation is attributed to the modulation of quenching efficiency. Quenching caused by fluorescence resonance energy transfer can lead to a nonlinear relationship between the modulation fluorescence strength and the applied ultrasound strength.

Simultaneous multiwavelength laminar optical tomography

Spatially resolved reflectance measurements can be used to characterize the depth-resolved optical properties of superficial tissues. However, until now, rapid acquisition of multiwavelength data has been hindered by multiplexing problems. We report on a novel multiwavelength laminar optical tomography system capable of acquiring data from multiple source-detector separations at three wavelengths simultaneously. Such data can allow in vivo depth-resolved spectroscopic imaging of absorbers, such as oxy- and deoxyhemoglobin, or of multiple fluorophores, that is unaffected by motion artifacts at frame rates exceeding 100 Hz. The system design and phantom validation studies are presented.

Separately reconstructing the structural and functional parameters of a fluorescent inclusion embedded in a turbid medium

We report a novel imaging technique for fluorescence diffuse optical tomography (FDOT). Unlike conventional FDOT, this technique separates the imaging procedure into two steps to respectively reconstruct the structural information (such as the center position and the radius), and the functional information (such as the fluorophore concentration and/or lifetime) of a fluorescing target embedded in a turbid medium. The structural parameters of the target were estimated from the amplitude ratio and phase difference of fluorescence signals received at different detectors, because the amplitude ratio and phase difference were found independent of, or weakly related to, the functional parameters. Based on the estimated structural parameters, a dual-zone mesh technique was utilized to reconstruct the fluorophore concentration. Results of simulations and phantom experiments showed that the structural parameters could be accurately recovered, without knowing the functional information, and that the reconstruction accuracy of the functional parameter was greater than 80%.

High-resolution imaging in a deep turbid medium based on an ultrasound-switchable fluorescence technique

The spatial resolution of fluorescence imaging techniques in deep optically turbid media such as tissues is limited by photon diffusion. To break the diffusion limit and achieve high-resolution and deep-tissue fluorescence imaging, a fundamentally different method was demonstrated based on a concept of ultrasound-switchable fluorescence. The results showed that a small fluorescent tube with a diameter of ∼180 μm at a depth of ∼20 mm in an optical scattering medium ([Formula: see text] and [Formula: see text] cm(-1)) can be clearly imaged with a size of ∼260 μm. The depth-to-resolution ratio is shown to be about one order of magnitude better than other deep-tissue fluorescence imaging techniques.

Ultrasound-modulated fluorescence from rhodamine B aqueous solution.

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.

Fluorescence imaging of vascular endothelial growth factor in tumors for mice embedded in a turbid medium

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.