Deep-tissue High-resolution Imaging Research Articles

Promoting the Near-Infrared-II Fluorescence of Diketopyrrolopyrrole-Based Dye for In Vivo Imaging via Donor Engineering.

Small-molecule dyes for fluorescence imaging in the second near-infrared region (NIR-II, 900-1880 nm) hold great promise in clinical applications. Constructing donor-acceptor-donor (D-A-D) architectures has been recognized to be a feasible strategy to achieve NIR-II fluorescence. However, the development of NIR-II dyes via such a scheme is hampered by the lack of high-performance electron acceptors and donors. Diketopyrrolopyrrole (DPP), as a classic organic optoelectronic material, enjoys strong light absorption, high fluorescence quantum yield (QY), and facile derivatization. Nevertheless, its application in the NIR-II imaging field has been hindered by its limited electron-withdrawing ability and the aggregation-caused quenching (ACQ) effect resulting from the planar structure of DPP. Herein, with DPP as an electron acceptor and through donor engineering, we have successfully designed and synthesized a DPP-based dye named T-27, in which the strong D-A interaction confers excellent NIR absorption and high-brightness NIR-II fluorescence tail emission. By strategically introducing long alkyl chains on the donor unit to increase intermolecular spacing and reduce the influence of solvent molecules, T-27 exhibits an improved anti-ACQ effect in aqueous solutions. After being encapsulated into DSPE-PEG2000, T-27 nanoparticles (NPs) show a relative NIR-II fluorescence QY of 3.4% in water, representing the highest value among the DPP-based NIR-II dyes reported to date. The outstanding photophysical properties of T-27 NPs enable multimode NIR-IIa bioimaging under 808 nm excitation. As such, the T-27 NPs can distinguish mouse femoral vein and artery and achieve cerebral vascular microscopic imaging with a penetrating depth of 800 μm, demonstrating the capability for high-resolution deep-tissue imaging. This work holds significant potential in the field of bioimaging and provides a new strategy for developing bright NIR-II dyes.

Noninvasive In Vivo Imaging of T-Cells during Cancer Immunotherapy Using Rare-Earth Nanoparticles.

Only a minority of patients respond positively to cancer immunotherapy, and addressing this variability is an active area of immunotherapy research. Infiltration of tumors by immune cells is one of the most significant prognostic indicators of response and disease-free survival. However, the ability to noninvasively sample the tumor microenvironment for immune cells remains limited. Imaging in the near-infrared-II region using rare-earth nanocrystals is emerging as a powerful imaging tool for high-resolution deep-tissue imaging. In this paper, we demonstrate that these nanoparticles can be used for noninvasive in vivo imaging of tumor-infiltrating T-cells in a highly aggressive melanoma tumor model. We present nanoparticle synthesis and surface modification strategies for the generation of small, ultrabright, and biocompatible rare-earth nanocrystals necessary for deep tissue imaging of rare cell types. The ability to noninvasively monitor the immune contexture of a tumor during immunotherapy could lead to early identification of nonresponding patients in real time, leading to earlier interventions and better outcomes.

Synthesis and luminescent properties of ZnWO4: Cr3+ nanoparticles for NIR-II fluorescence imaging

Fluorescence imaging is of paramount importance in the health and medical aspect, especially for emission in second near-infrared window, where high-resolution deep-tissue imaging can be achieved. Herein, ZnWO4: Cr3+ nanoparticles (NPs) with broadband NIR-II emission were synthesized by a facile solvothermal+calcination method. It is found that Cr3+-doping have no obvious effect of ZnWO4 phase, and the particles of as-prepared NPs are of mono-dispersed cube-shape with average size of ∼100 nm. At optimal 520 nm excitation, the as-synthesized ZnWO4: Cr3+ NPs exhibit an efficient NIR emission peaking at 987 nm and EQE of 4.03% with FWHM of 212 nm. The ZnWO4: Cr3+ NPs possess excellent stability, good biocompatibility and high brightness. NIR-II imaging with high penetration depth and micron spatial resolution was achieved on a mice model. To sum up, our work indicates that ZnWO4: Cr3+ NPs has great application potential as fluorescent agent for NIR-II bio-imaging.

Coaxial interferometry for camera-based ultrasound-modulated optical tomography with paired illumination.

Ultrasound-modulated optical tomography (UOT), which combines the advantages of both light and ultrasound, is a promising imaging modality for deep-tissue high-resolution imaging. Among existing implementations, camera-based UOT gains huge advances in modulation depth through parallel detection. However, limited by the long exposure time and the slow framerate of modern cameras, the measurement of UOT signals always requires holographic methods with additional reference beams. This requirement increases system complexity and is susceptible to environmental disturbances. To overcome this challenge, we develop coaxial interferometry for camera-based UOT in this work. Such a coaxial scheme is enabled by employing paired illumination with slightly different optical frequencies. To measure the UOT signal, the conventional phase-stepping method in holography can be directly transplanted into coaxial interferometry. Specifically, we performed both numerical investigations and experimental validations for camera-based UOT under the proposed coaxial scheme. One-dimensional imaging for an absorptive target buried inside a scattering medium was demonstrated. With coaxial interferometry, this work presents an effective way to reduce system complexity and cope with environmental disturbances for camera-based UOT.

Looking deep inside tissue with photoacoustic molecular probes: a review.

.SignificanceDeep tissue noninvasive high-resolution imaging with light is challenging due to the high degree of light absorption and scattering in biological tissue. Photoacoustic imaging (PAI) can overcome some of the challenges of pure optical or ultrasound imaging to provide high-resolution deep tissue imaging. However, label-free PAI signals from light absorbing chromophores within the tissue are nonspecific. The use of exogeneous contrast agents (probes) not only enhances the imaging contrast (and imaging depth) but also increases the specificity of PAI by binding only to targeted molecules and often providing signals distinct from the background.AimWe aim to review the current development and future progression of photoacoustic molecular probes/contrast agents.ApproachFirst, PAI and the need for using contrast agents are briefly introduced. Then, the recent development of contrast agents in terms of materials used to construct them is discussed. Then, various probes are discussed based on targeting mechanisms, in vivo molecular imaging applications, multimodal uses, and use in theranostic applications.ResultsMaterial combinations are being used to develop highly specific contrast agents. In addition to passive accumulation, probes utilizing activation mechanisms show promise for greater controllability. Several probes also enable concurrent multimodal use with fluorescence, ultrasound, Raman, magnetic resonance imaging, and computed tomography. Finally, targeted probes are also shown to aid localized and molecularly specific photo-induced therapy.ConclusionsThe development of contrast agents provides a promising prospect for increased contrast, higher imaging depth, and molecularly specific information. Of note are agents that allow for controlled activation, explore other optical windows, and enable multimodal use to overcome some of the shortcomings of label-free PAI.

A Review of Endogenous and Exogenous Contrast Agents Used in Photoacoustic Tomography with Different Sensing Configurations.

Optical-based sensing approaches have long been an indispensable way to detect molecules in biological tissues for various biomedical research and applications. The advancement in optical microscopy is one of the main drivers for discoveries and innovations in both life science and biomedical imaging. However, the shallow imaging depth due to the use of ballistic photons fundamentally limits optical imaging approaches’ translational potential to a clinical setting. Photoacoustic (PA) tomography (PAT) is a rapidly growing hybrid imaging modality that is capable of acoustically detecting optical contrast. PAT uniquely enjoys high-resolution deep-tissue imaging owing to the utilization of diffused photons. The exploration of endogenous contrast agents and the development of exogenous contrast agents further improve the molecular specificity for PAT. PAT’s versatile design and non-invasive nature have proven its great potential as a biomedical imaging tool for a multitude of biomedical applications. In this review, representative endogenous and exogenous PA contrast agents will be introduced alongside common PAT system configurations, including the latest advances of all-optical acoustic sensing techniques.

Transparent capacitive micromachined ultrasonic transducer (CMUT) arrays for real-time photoacoustic applications.

Photoacoustic imaging has shown great potential for non-invasive high-resolution deep-tissue imaging. Minimizing the optical and acoustic paths for excitation and detection could significantly increase the signal-to-noise ratio. This could be accomplished by transparent transducers permitting through-transducer illumination. However, most ultrasound transducers are not optically transparent. Capacitive micromachined ultrasound transducer (CMUT) technology has compelling properties compared to piezoelectric transducers such as wide bandwidth and high receive sensitivity. Here, we introduce transparent CMUT linear arrays with high transparency in the visible and near-infrared range. To fabricate the devices, we used an adhesive wafer bonding technique using photosensitive benzocyclobutene (BCB) as both a structural and adhesive layer with a glass-indium-tin-oxide (ITO) substrate. Silicon nitride is used as the membrane material ensuring hermiticity and optical transparency. Our fabricated transducer arrays consist of 64 and 128 elements with immersion operation frequency of 8 MHz, enabling high-resolution imaging. ITO, along with thin metal strips, are used as a conductive layer for the top electrodes with minimal impact on device transparency. Fabricated devices have shown average transparency of 70% in the visible wavelength range that goes up to 90% in the near-infrared range. Arrays are wire-bonded to interfacing electronics and connected to a research ultrasound platform for phantom imaging. Arrays exhibited signal-to-noise (SNR) of 40 dB with 30V bias voltage and laser fluence of 13.5 mJ/cm2. Arrays with 128 channels provided lateral and axial resolutions of 234 µm and 220 µm, respectively.

Two-Photon Dual-Emissive Carbon Dot-Based Probe: Deep-Tissue Imaging and Ultrasensitive Sensing of Intracellular Ferric Ions.

Carbon dots (CDs)-based nanoparticles have been extensively explored for biological applications in sensing and bioimaging. However, the major translational barriers to CDs for imaging and sensing applications include synthetic strategies to obtain monodisperse CDs with tunable structural, electronic, and optical properties in order to achieve high-resolution deep-tissue imaging, intracellular detection, and sensing of metal ions with high sensitivity down to nanomolar levels. Herein, we report a novel strategy to synthesize and develop a multifunctional nitrogen-doped CDs probe of different sizes using a new combination of carbon and nitrogen sources. Our results show that the structural characteristics (i.e., the surface density of emissive traps and bandgaps levels) depend on the size of the CDs, which ultimately influences their optical properties. This work also demonstrates the development of a two-photon dual-emissive fluorescent multifunctional probes (3-FCDs) by conjugating fluorescein isothiocyanate on the surface of nitrogen-doped CDs. 3-FCDs show excellent near-infrared two-photon excitation ability, single-wavelength excitation, high photostability, biocompatibility, low cytotoxicity, and good cell permeability. Using two-photon fluorescence imaging, our multifunctional probe shows excellent deep-tissue high-resolution imaging capabilities with penetration depth up to 3000 and 280 μm in hydrogel scaffold and pigskin tissue, respectively. The designed probe exhibits ultrasensitivity and specificity toward Fe3+ ions with a remarkable detection limit of 2.21 nM using two-photon excitation. In addition, we also demonstrate the use of multifunctional CDs probe for ultrasensitive exogenous and real-time endogenous sensing of Fe3+ ions and imaging in live fibroblasts with rapid response times for intracellular ferric ion detection.

3D microphotonic probe for high resolution deep tissue imaging.

Ultra-compact miniaturized optical components for microendoscopic tools and miniaturized microscopes are required for minimally invasive imaging. Current microendoscopic technologies used for deep tissue imaging procedures are limited to a large diameter and/or low resolution due to manufacturing restrictions. We demonstrate a platform for miniaturization of an optical imaging system for microendoscopic applications with a resolution of 1 µm. We designed our probe using cascaded micro-lenses and waveguides (lensguide) to achieve a probe as small as 100 µm x 100 µm with a field of view of 60 µm in diameter. We demonstrate wide-field microscopy based on our polymeric probe fabricated using photolithography and a two-photon polymerization process.

Miniature all-fiber axicon probe with extended Bessel focus for optical coherence tomography.

The trade-off between lateral resolution and depth of focus (DOF) severely limits the capability of endoscopic optical coherence tomography (OCT) for high-resolution deep-tissue imaging. To address this issue, we developed a novel miniature all-fiber axicon OCT probe by inserting a segment of gradient-index (GRIN) fiber between a piece of single-mode fiber (SMF) and an axicon polished from a no-core fiber. The GRIN lens served as a beam expander extending the probe DOF by 5.2 times while maintaining a high lateral resolution of 2 μm. The DOF extension was experimentally verified by measuring the axial profile of the probe output beam and further by imaging multilayered polymer tapes and onion samples. The designed probe with a tight focus over a large DOF holds great potential in endoscopic OCT imaging of deep tissues at the cellular level.

Modulation of ultrasound-switchable fluorescence for improving signal-to-noise ratio.

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.

Label-free photoacoustic tomography of whole mouse brain structures ex vivo.

Capitalizing on endogenous hemoglobin contrast, photoacoustic-computed tomography (PACT), a deep-tissue high-resolution imaging modality, has drawn increasing interest in neuroimaging. However, most existing studies are limited to functional imaging on the cortical surface and the deep brain structural imaging capability of PACT has never been demonstrated. Here, we explicitly studied the limiting factors of deep brain PACT imaging. We found that the skull distorted the acoustic signal and blood suppressed the structural contrast from other chromophores. When the two effects are mitigated, PACT can potentially provide high-resolution label-free imaging of structures in the entire mouse brain. With [Formula: see text] in-plane resolution, we can clearly identify major structures of the brain, which complements magnetic resonance microscopy for imaging small-animal brain structures. Spectral PACT studies indicate that structural contrasts mainly originate from cytochrome distribution and that the presence of lipid sharpens the image contrast; brain histology results provide further validation. The feasibility of imaging the structure of the brain in vivo is also discussed. Our results demonstrate that PACT is a promising modality for both structural and functional brain imaging.

Fluorescent Cell Imaging in Regenerative Medicine

Fluorescent protein imaging, a promising tool in biological research, incorporates numerous applications that can be of specific use in the field of regenerative medicine. To enhance tissue regeneration efforts, scientists have been developing new ways to monitor tissue development and maturation in vitro and in vivo. To that end, new imaging tools and novel fluorescent proteins have been developed for the purpose of performing deep-tissue high-resolution imaging. These new methods, such as intra-vital microscopy and Förster resonance energy transfer, are providing new insights into cellular behavior, including cell migration, morphology, and phenotypic changes in a dynamic environment. Such applications, combined with multimodal imaging, significantly expand the utility of fluorescent protein imaging in research and clinical applications of regenerative medicine.

Imaging analysis of insulin secretion with two-photon microscopy.

High-resolution deep tissue imaging is possible with two-photon excitation microscopy. With the combined application of two-photon imaging and perfusion with a polar fluorescent tracer, we have established a method to detect exocytic events inside secretory tissues. This method displays the spatiotemporal distribution of exocytic sites, dynamics of fusion pores, and modes of exocytosis. In glucose-stimulated pancreatic islets, exocytic events were observed to be synchronized with an increase in cytosolic Ca(2+) concentrations. Full fusion of a single secretory granule is the typical mode of exocytosis and compound exocytosis is inhibited. Because two-photon excitation enables simultaneous multicolor imaging due to the broadened excitation spectra, the distributions and conformational changes in fluorescent-labeled molecules can be simultaneously visualized with exocytic events. Therefore, we can analyze the dynamics of the molecules involved in membrane fusion and their association with exocytosis in living tissues.

Nonlinear photoacoustic microscopy via a loss modulation technique: from detection to imaging

In order to achieve high-resolution deep-tissue imaging, multi-photon fluorescence microscopy and photoacoustic tomography had been proposed in the past two decades. However, combining the advantages of these two imaging systems to achieve optical-spatial resolution with an ultrasonic-penetration depth is still a field with challenges. In this paper, we investigate the detection of the two-photon photoacoustic ultrasound, and first demonstrate background-free two-photon photoacoustic imaging in a phantom sample. To generate the background-free two-photon photoacoustic signals, we used a high-repetition rate femtosecond laser to induce narrowband excitation. Combining a loss modulation technique, we successfully created a beating on the light intensity, which not only provides pure sinusoidal modulation, but also ensures the spectrum sensitivity and frequency selectivity. By using the lock-in detection, the power dependency experiment validates our methodology to frequency-select the source of the nonlinearity. This ensures our capability of measuring the background-free two-photon photoacoustic waves by detecting the 2nd order beating signal directly. Furthermore, by mixing the nanoparticles and fluorescence dyes as contrast agents, the two-photon photoacoustic signal was found to be enhanced and detected. In the end, we demonstrate subsurface two-photon photoacoustic bio-imaging based on the optical scanning mechanism inside phantom samples.

Breaking the acoustic diffraction limit via nonlinear effect and thermal confinement for potential deep-tissue high-resolution imaging

Our recently developed ultrasound-switchable fluorescence (USF) imaging technique showed that it was feasible to conduct high-resolution fluorescence imaging in a centimeter-deep turbid medium. Because the spatial resolution of this technique highly depends on the ultrasound-induced temperature focal size (UTFS), minimization of UTFS becomes important for further improving the spatial resolution USF technique. In this study, we found that UTFS can be significantly reduced below the diffraction-limited acoustic intensity focal size via nonlinear acoustic effects and thermal confinement by appropriately controlling ultrasound power and exposure time, which can be potentially used for deep-tissue high-resolution imaging.

Non-linear fluorescence lifetime imaging of biological tissues

In recent years fluorescence microscopy has become a widely used tool for tissue imaging and spectroscopy. Optical techniques, based on both linear and non-linear excitation, have been broadly applied to imaging and characterization of biological tissues. Among fluorescence techniques used in tissue imaging applications, in recent years two and three-photon excited fluorescence have gained increased importance because of their high-resolution deep tissue imaging capability inside optically turbid samples. The main limitation of steady-state fluorescence imaging techniques consists in providing only morphological information; functional information is not detectable without technical improvements. A spectroscopic approach, based on lifetime measurement of tissue fluorescence, can provide functional information about tissue conditions, including its environment, red-ox state, and pH, and hence physiological characterization of the tissue under investigation. Measurement of the fluorescence lifetime is a very important issue for characterizing a biological tissue. Deviation of this property from a control value can be taken as an indicator of disorder and/or malignancy in diseased tissues. Even if much work on this topic has still to be done, including the interpretation of fluorescence lifetime data, we believe that this methodology will gain increasing importance in the field of biophotonics. In this paper, we review methodologies, potentials and results obtained by using fluorescence lifetime imaging microscopy for the investigation of biological tissues.