Ultrasound-switchable Fluorescence Imaging Research Articles

Improving the spatial resolution and signal-to-noise ratio of ultrasound switchable fluorescence imaging.

Ultrasound switchable fluorescence (USF) imaging, a hybrid imaging technology that combines the advantages of both fluorescence sensitivity and acoustic resolution in centimeter-deep tissue, has great potential for biomedical different applications. A camera-based USF imaging system reveals its capability of capturing both spatial and temporal dynamics of the USF signal in tissue. In this study, various algorithms were explored to enhance the spatial resolution and signal-to-noise ratio (SNR) of USF images, utilizing temporal and spatial information from a camera-based time-domain USF imaging system. The correlation method proved effective in boosting SNR, while the ascending-slope-weighted method enhanced spatial resolution. Additionally, the spatially back-projection method significantly improved spatial resolution in silicone phantoms. The results underscore the advantages of incorporating temporal and spatial information from USF signals.

Temperature-sensitive polymeric nanogels encapsulating with β-cyclodextrin and ICG complex for high-resolution deep-tissue ultrasound-switchable fluorescence imaging

One of the thorny problems currently impeding the applications of the fluorescence imaging technique is the poor spatial resolution in deep tissue. Ultrasound-switchable fluorescence (USF) imaging is a novel imaging tool that has recently been explored to possibly surmount the above-mentioned bottleneck. Herein, a β-cyclodextrin/indocyanine green (ICG) complex-encapsulated poly(N-isopropylacrylamide) (PNIPAM) nanogel was synthesized and studied for ex vivo/in vivo deep tissue/high-resolution near infrared USF (NIR-USF) imaging. To be specific, our results revealed that the average diameter of the as-prepared nanogels was significantly decreased to ~ 32 nm from ~ 335 nm compared to the reported ICG-PNIPAM nanoparticles. Additionally, the excitation/ emission characteristics of the ICG itself in present nanogels were almost completely retained, and the resultant nanogel exhibited high physiological stability and positive biocompatibility. In particular, the signal-to-noise ratio of the USF image for the PNIPAM/ β-cyclodextrin/ICG nanogel (33.01 ± 2.42 dB) was prominently higher than that of the ICG-PNIPAM nanoparticles (18.73 ± 0.33 dB) in 1.5-cm-thick chicken breast tissues. The NIR-USF imaging in 3.5-cm-thick chicken breast tissues was achieved using this new probe. The ex vivo NIR-USF imaging of the mouse liver was also successfully obtained. Animal experiments showed that the present nanogels were able to be effectively accumulated into U87 tumor-bearing mice via enhanced permeability and retention effects, and the high-resolution NIR-USF imaging of in vivo tumor was efficiently acquired. The metabolism and in vivo biodistribution of the nanogels were evaluated. Overall, the results suggest that the current nanogel is a highly promising NIR-USF probe for deep tissue and high-resolution USF imaging.

Improving sensitivity and imaging depth of ultrasound-switchable fluorescence via an EMCCD-gain-controlled system and a liposome-based contrast agent.

The ultrasound-switchable fluorescence (USF) technique was recently developed to achieve high-resolution fluorescence imaging in centimeters-deep tissue. This study introduced strategies to significantly improve imaging sensitivity and depth using an electron multiplying charge-coupled device (EMCCD) camera-based USF imaging system and a newly developed USF contrast agent of indocyanine green (ICG)-encapsulated liposomes. For a quantitative study, a phantom of a sub-millimeter silicone tube embedded in centimeter-thick chicken breast tissue was adopted in this study as a model. The synthesized ICG-liposome was characterized and compared with the previously reported ICG-nanogel. The exposure of the EMCCD camera was controlled via the MATLAB (The MathWorks, Inc. USA), instead of an external hardware trigger. The stability of the electron multiplying (EM) gain of the EMCCD camera was compared between two trigger modes: the MATLAB trigger mode and the external hardware trigger mode. The signal-to-noise ratio (SNR) of the USF imaging with different EM gain in various thick tissue was studied. The hydrodynamic size of the ICG-liposome was ~181 nm. The ICG-liposome had a sharper temperature switching curve and a better USF performance than the previously reported ICG-nanogel. The EM gain was more stable in MATLAB trigger mode than the external hardware trigger mode. Although, as usual, the SNR decreased quickly with the increase of the tissue thickness, the proposed strategies improved the SNR and the imaging depth significantly by adopting the novel contrast agent and controlling the EM gain. We successfully imaged the sub-millimeter silicone tube with an inner diameter of 0.76 mm and an outer diameter of 1.65 mm in 5.5 cm-thick chicken breast tissue using 808 nm excitation light with a low intensity of 28.35 mW/cm2, the improved EMCCD camera-based USF imaging system and the novel ICG-liposomes.

In vivo ultrasound-switchable fluorescence imaging using a camera-based system.

Ultrasound-switchable fluorescence (USF) is a novel imaging technique that provides high spatial resolution fluorescence images in centimeter-deep biological tissue. Recently, we successfully demonstrated the feasibility of in vivo USF imaging using a frequency-domain photomultiplier tube-based system. In this work, for the first time we carried out in vivo USF imaging via a camera-based USF imaging system. The system acquires a USF signal on a two-dimensional (2D) plane, which facilitates the image acquisition because the USF scanning area can be planned based on the 2D image and provides high USF photon collection efficiency. We demonstrated in vivo USF imaging in the mouse's glioblastoma tumor with multiple targets via local injection. In addition, we designed the USF contrast agents with different particle sizes (70 nm and 330 nm) so that they could bio-distribute to various organs (spleen, liver, and kidney) via intravenous (IV) injections. The results showed that the contrast agents retained stable USF properties in tumors and some organs (spleen and liver). We successfully achieved in vivo USF imaging of the mouse's spleen and liver via IV injections. The USF imaging results were compared with the images acquired from a commercial X-ray micro computed tomography (micro-CT) system.

A Biocompatible and Near‐Infrared Liposome for In Vivo Ultrasound‐Switchable Fluorescence Imaging

Fluorescence imaging is a remarkable tool for molecular targeting and multicolor imaging, but it suffers from low resolution in centimeter-deep tissues. The recently developed ultrasound-switchable fluorescence (USF) imaging has overcome this challenge and achieved in vivo imaging in a mouse with help from the indocyanine green (ICG) dye encapsulated poly(N-isopropylacrylamide) (ICG-PNIPAM) contrast agent. However, the ICG-PNIPAM has shortcomings, such as concerns about cytotoxicity and blueshifted excitation and emission spectra. This study introduces a newly developed ICG-encapsulated liposome to broaden the contrast agent selection for USF imaging and resolve the issues mentioned above. The emission peak of the ICG-liposome is 836 nm with excellent biostability and USF imaging capability. Furthermore, the cell viability test verifies the low cytotoxicity feature. Eventually, both ex vivo and in vivo USF imaging are successfully achieved and 3D USF images are acquired. The ex vivo result confirms that the ICG-liposome maintains the thermoresponsive characteristic at the right lobe of the liver and is able to conduct the USF imaging. The further in vivo USF imaging demonstrates that although the whole liver emitted fluorescence, only the right lobe of the liver contains the working ICG-liposome.

An ICCD camera-based time-domain ultrasound-switchable fluorescence imaging system

Fluorescence imaging in centimeter-deep tissues with high resolution is highly desirable for many biomedical applications. Recently, we have developed a new imaging modality, ultrasound-switchable fluorescence (USF) imaging, for achieving this goal. In our previous work, we successfully achieved USF imaging with several types of USF contrast agents and imaging systems. In this study, we introduced a new USF imaging system: an intensified charge-coupled device (ICCD) camera-based, time-domain USF imaging system. We demonstrated the principle of time-domain USF imaging by using two USF contrast agents. With a series of USF imaging experiments, we demonstrated the tradeoffs among different experimental parameters (i.e., data acquisition time, including CCD camera recording time and intensifier gate delay; focused ultrasound (FU) power; and imaging depth) and the image qualities (i.e., signal-to-noise ratio, spatial resolution, and temporal resolution). In this study, we also discussed several imaging strategies for achieving a high-quality USF image via this time-domain system.

In vivo ultrasound-switchable fluorescence imaging

The conventional fluorescence imaging has limited spatial resolution in centimeter-deep tissue because of the tissue’s high scattering property. Ultrasound-switchable fluorescence (USF) imaging, a new imaging technique, was recently proposed to realize high-resolution fluorescence imaging in centimeter-deep tissue. However, in vivo USF imaging has not been achieved so far because of the lack of stable near-infrared contrast agents in a biological environment and the lack of data about their biodistributions. In this study, for the first time, we achieved in vivo USF imaging successfully in mice with high resolution. USF imaging in porcine heart tissue and mouse breast tumor via local injections were studied and demonstrated. In vivo and ex vivo USF imaging of the mouse spleen via intravenous injections was also successfully achieved. The results showed that the USF contrast agent adopted in this study was very stable in a biological environment, and it was mainly accumulated into the spleen of the mice. By comparing the results of CT imaging and the results of USF imaging, the accuracy of USF imaging was proved.

Ultrasound-Switchable Fluorescence Imaging via an EMCCD Camera and a Z-Scan Method

The spatial resolution of the conventional fluorescence imaging in centimeter-deep tissue is limited due to high light scattering. Ultrasound-switchable fluorescence (USF) imaging was recently developed to achieve high resolution in deep tissue. However, previous frequency-domain USF systems adopted a fiber bundle to collect the emitted fluorescence photons, which led to a few limitations, such as, first, low photon-collection efficiency; second, low efficiency for scanning a sample with an uneven surface; third, low imaging speed, because of the adoption of a raster scan and the long wait time for the tissue to cool down before starting the data acquisition at the next location. In this study, we proposed a camera-based USF imaging system to overcome these limitations. Thanks to the spatial information provided by the camera, a new scan method, Z-scan, was developed, and the imaging speed was improved four times over the raster scan. The USF imaging using the Z-scan for tissue samples was realized. The results were validated by a commercial micro-CT system.

A Dual-Modality System for Both Multi-Color Ultrasound-Switchable Fluorescence and Ultrasound Imaging.

Simultaneous imaging of multiple targets (SIMT) in opaque biological tissues is an important goal for molecular imaging in the future. Multi-color fluorescence imaging in deep tissues is a promising technology to reach this goal. In this work, we developed a dual-modality imaging system by combining our recently developed ultrasound-switchable fluorescence (USF) imaging technology with the conventional ultrasound (US) B-mode imaging. This dual-modality system can simultaneously image tissue acoustic structure information and multi-color fluorophores in centimeter-deep tissue with comparable spatial resolutions. To conduct USF imaging on the same plane (i.e., x-z plane) as US imaging, we adopted two 90°-crossed ultrasound transducers with an overlapped focal region, while the US transducer (the third one) was positioned at the center of these two USF transducers. Thus, the axial resolution of USF is close to the lateral resolution, which allows a point-by-point USF scanning on the same plane as the US imaging. Both multi-color USF and ultrasound imaging of a tissue phantom were demonstrated.

New generation ICG-based contrast agents for ultrasound-switchable fluorescence imaging.

Recently, we developed a new technology, ultrasound-switchable fluorescence (USF), for high-resolution imaging in centimeter-deep tissues via fluorescence contrast. The success of USF imaging highly relies on excellent contrast agents. ICG-encapsulated poly(N-isopropylacrylamide) nanoparticles (ICG-NPs) are one of the families of the most successful near-infrared (NIR) USF contrast agents. However, the first-generation ICG-NPs have a short shelf life (<1 month). This work significantly increases the shelf life of the new-generation ICG-NPs (>6 months). In addition, we have conjugated hydroxyl or carboxyl function groups on the ICG-NPs for future molecular targeting. Finally, we have demonstrated the effect of temperature-switching threshold (Tth) and the background temperature (TBG) on the quality of USF images. We estimated that the Tth of the ICG-NPs should be controlled at ~38–40 °C (slightly above the body temperature of 37 °C) for future in vivo USF imaging. Addressing these challenges further reduces the application barriers of USF imaging.

Development of Ultrasound-switchable Fluorescence Imaging Contrast Agents based on Thermosensitive Polymers and Nanoparticles.

In this work we first introduced a recently developed high-resolution, deep-tissue imaging technique, ultrasound-switchable fluorescence (USF). The imaging principles based on two types of USF contrast agents were reviewed. To improve USF imaging techniques further, excellent USF contrast agents were developed based on high-performance thermoresponsive polymers and environment-sensitive fluorophores. Herein, such contrast agents were synthesized and characterized with five key parameters: (1) peak excitation and emission wavelengths (λex and λem), (2) the fluorescence intensity ratio between on and off states (IOn/IOff), (3) the fluorescence lifetime ratio between on and off states (τOn/τOff), (4) the temperature threshold to switch on fluorophores (Tth), and (5) the temperature transition bandwidth (TBW). We mainly investigated fluorescence intensity and lifetime changes of four environment-sensitive dyes [7-(2-Aminoethylamino)-N,N-dimethyl-4-benzofurazansulfonamide (DBD-ED), St633, Sq660, and St700] as a function of temperature, while the dye was attached to poly(N-isopropylacrylamide) linear polymers or encapsulated in nanoparticles. Six fluorescence resonance energy transfer systems were invented in which both the donor (DBD-ED or ST425) and the acceptor (Sq660) were adopted. Our results indicate that three Förster resonance energy transfer systems, where both IOn/IOff and τOn/τOff are larger than 2.5, are promising for application in future surface tissue bioimaging by USF technique.

High resolution imaging beyond the acoustic diffraction limit in deep tissue via ultrasound-switchable NIR fluorescence.

Fluorescence imaging in deep tissue with high spatial resolution is highly desirable because it can provide details about tissue's structural, functional, and molecular information. Unfortunately, current fluorescence imaging techniques are limited either in penetration depth (microscopy) or spatial resolution (diffuse light based imaging) as a result of strong light scattering in deep tissue. To overcome this limitation, we developed an ultrasound-switchable fluorescence (USF) imaging technique whereby ultrasound was used to switch on/off the emission of near infrared (NIR) fluorophores. We synthesized and characterized unique NIR USF contrast agents. The excellent switching properties of these agents, combined with the sensitive USF imaging system developed in this study, enabled us to image fluorescent targets in deep tissue with spatial resolution beyond the acoustic diffraction limit.