Deep-tissue Optical Imaging Research Articles

Efficient and Stable NIR-II Phosphorescence of Metallophilic Molecular Oligomers for In Vivo Single-Cell Tracking and Time-Resolved Imaging.

Molecular phosphorescence in the second near-infrared window (NIR-II, 1000-1700 nm) holds promise for deep-tissue optical imaging with high contrast by overcoming background fluorescence interference. However, achieving bright and stable NIR-II molecular phosphorescence suitable for biological applications remains a formidable challenge. Herein, we report a new series of symmetric isocyanorhodium(I) complexes that could form oligomers and exhibit bright, long-lived (7-8 μs) phosphorescence in aqueous solution via metallophilic interaction. Ligand substituents with enhanced dispersion attraction and electron-donating properties were explored to extend excitation/emission wavelengths and enhanced stability. Further binding the oligomers with fetal bovine serum (FBS) resulted in NIR-II molecular phosphorescence with high quantum yields (up to 3.93 %) and long-term stability in biological environments, enabling in vivo tracking of single-macrophage dynamics and high-contrast time-resolved imaging. These results pave the way for the development of highly-efficient NIR-II molecular phosphorescence for biomedical applications.

Unravelling Immune-Inflammatory Responses and Lysosomal Adaptation: Insights from Two-Photon Excited Delayed Fluorescence Imaging.

Two-photon excitation (TPE) microscopy with near-infrared (NIR) emission has emerged as a promising technique for deep-tissue optical imaging. Recent developments in fluorescence lifetime imaging with long-lived emission probes have further enhanced the spatial resolution and precision of fluorescence imaging, especially in complex systems with short-lived background signals. In this study, two innovative lysosome-targeting probes, Cz-NA and tCz-NA, are introduced. These probes offer a combination of advantages, including TPE (λex = 880nm), NIR emission (λem = 650nm), and thermally activated delayed fluorescence (TADF) with long-lived lifetimes (1.05 and 1.71 µs, respectively). These characteristics significantly improve the resolution and signal-to-noise ratio in deep-tissue imaging. By integrating an acousto-optic modulator (AOM) device with TPE microscopy, the authors successfully applied Cz-NA in two-photon excited delayed fluorescence (TPEDF) imaging to track lysosomal adaptation and immune responses to inflammation in mice. This study sheds light on the relationship between lysosome tubulation, innate immune responses, and inflammation in vivo, providing valuable insights for the development of autofluorescence-free molecular probes in the future.

Chemiluminescent Conjugated Polymer Nanoparticles for Deep-Tissue Inflammation Imaging and Photodynamic Therapy of Cancer.

Deep-tissue optical imaging and photodynamic therapy (PDT) remain a big challenge for the diagnosis and treatment of cancer. Chemiluminescence (CL) has emerged as a promising tool for biological imaging and in vivo therapy. The development of covalent-binding chemiluminescence agents with high stability and high chemiluminescence resonance energy transfer (CRET) efficiency is urgent. Herein, we design and synthesize an unprecedented chemiluminescent conjugated polymer PFV-Luminol, which consists of conjugated polyfluorene vinylene (PFV) main chains and isoluminol-modified side chains. Notably, isoluminol groups with chemiluminescent ability are covalently linked to main chains by amide bonds, which dramatically narrow their distance, greatly improving the CRET efficiency. In the presence of pathologically high levels of various reactive oxygen species (ROS), especially singlet oxygen (1O2), PFV-Luminol emits strong fluorescence and produces more ROS. Furthermore, we construct the PFV-L@PEG-NPs and PFV-L@PEG-FA-NPs nanoparticles by self-assembly of PFV-Luminol and amphiphilic copolymer DSPE-PEG/DSPE-PEG-FA. The chemiluminescent PFV-L@PEG-NPs nanoparticles exhibit excellent capabilities for in vivo imaging in different inflammatory animal models with great tissue penetration and resolution. In addition, PFV-L@PEG-FA-NPs nanoparticles show both sensitive in vivo chemiluminescence imaging and efficient chemiluminescence-mediated PDT for antitumors. This study paves the way for the design of chemiluminescent probes and their applications in the diagnosis and therapy of diseases.

Lanthanide doped NaYF4 core@multi-shell nanoparticles: Synthetic strategy for emission in divergent spectral regions

We present the use of lanthanide doped NaYF4 based core@multiple-shell nanoparticles as optical materials showing merged possibilities of engineered photon management processes. We have successfully synthesized and characterized series of NaYF4 core@shell@shell@shell nanomaterials in which the core parts were doped with NIR absorbing/emitting lanthanides (i.e., Nd3+, Yb3+, and Tm3+) to benefit from the complete separation of peristatic quenching processes connected to the interaction with solvent and/or ligand molecules. Followed by covering with an inert shell, Pr3+ ions were doped for VIS-to-UVC up-conversion in the next layer , and finally covered with yet another un-doped protecting shell. The obtained materials were characterized by the means of morphology, crystal structure and spectroscopic properties, with the emphasis put on the measurements in the UVC and NIR spectral regions. The obtained results showed the interesting possibility of merging within designed nanoparticles architectures both UVC light generation for future therapeutic purposes and NIR emission for deep tissue optical imaging diagnostics.

Deep-tissue photoacoustic imaging of photoswitchable transgenic mouse models

Compared with visible light, near-infrared (NIR) light (700–1300 nm) is capable of deep-tissue penetration because of low tissue attenuation, and thus holds great promise for deep-tissue optical manipulation and imaging. In this work, we take advantages of the NIR Rhodopseudomonas palustris bacterial phytochrome photoreceptors (BphP1) and generate a loxP-BphP1 photoswitchable transgenic mouse model that enables Cre-dependent temporal and spatial targeting of BphP1 expression at the whole-body scale in vivo. This BphP1 phytochrome incorporates biliverdin chromophore and reversibly switches between the ground state and activated state, which enabled the light-controlled manipulation of gene expression and differential detection in photoacoustic tomography (PAT). We first validated the optogenetic performance of BphP1 that binds its engineered protein partner QPAS1 to trigger gene transcription in primary cells and living mice, and demonstrated its superior capability of deep-tissue optogenetics over the existing visible-light proteins. Then, by taking advantage of PAT's deep-tissue non-invasive imaging ability and the BphP1's photoswitching properties, we can suppress the non-switching signals from background blood and improve the molecular detection sensitivity by three orders of magnitude. This study shows the BphP1-encoded photoswitchable transgenic mouse model can be used for both NIR optogenetic manipulation and deep-tissue PAT.

Snapshot time-reversed ultrasonically encoded optical focusing guided by time-reversed photoacoustic wave

Deep-tissue optical imaging is a longstanding challenge limited by scattering. Both optical imaging and treatment can benefit from focusing light in deep tissue beyond one transport mean free path. Wavefront shaping based on time-reversed ultrasonically encoded (TRUE) optical focusing utilizes ultrasound focus, which is much less scattered than light in biological tissues as the 'guide star'. However, the traditional TRUE is limited by the ultrasound focusing area and pressure tagging efficiency, especially in acoustically heterogeneous medium. Even the improved version of iterative TRUE comes at a large time consumption, which limits the application of TRUE. To address this problem, we proposed a method called time-reversed photoacoustic wave guided time-reversed ultrasonically encoded (TRPA-TRUE) optical focusing by integrating accurate ultrasonic focusing through acoustically heterogeneous medium guided by time-reversing PA signals, and the ultrasound modulation of diffused coherent light with optical phase conjugation (OPC), achieving dynamic focusing of light into scattering medium. Simulation results show that the focusing accuracy of the proposed method has been significantly improved compared with conventional TRUE, which is more suitable for practical applications that suffers severe acoustic distortion, e.g. transcranial optical focusing.

High-throughput volumetric adaptive optical imaging using compressed time-reversal matrix

Deep-tissue optical imaging suffers from the reduction of resolving power due to tissue-induced optical aberrations and multiple scattering noise. Reflection matrix approaches recording the maps of backscattered waves for all the possible orthogonal input channels have provided formidable solutions for removing severe aberrations and recovering the ideal diffraction-limited spatial resolution without relying on fluorescence labeling and guide stars. However, measuring the full input–output response of the tissue specimen is time-consuming, making the real-time image acquisition difficult. Here, we present the use of a time-reversal matrix, instead of the reflection matrix, for fast high-resolution volumetric imaging of a mouse brain. The time-reversal matrix reduces two-way problem to one-way problem, which effectively relieves the requirement for the coverage of input channels. Using a newly developed aberration correction algorithm designed for the time-reversal matrix, we demonstrated the correction of complex aberrations using as small as 2% of the complete basis while maintaining the image reconstruction fidelity comparable to the fully sampled reflection matrix. Due to nearly 100-fold reduction in the matrix recording time, we could achieve real-time aberration-correction imaging for a field of view of 40 × 40 µm2 (176 × 176 pixels) at a frame rate of 80 Hz. Furthermore, we demonstrated high-throughput volumetric adaptive optical imaging of a mouse brain by recording a volume of 128 × 128 × 125 µm3 (568 × 568 × 125 voxels) in 3.58 s, correcting tissue aberrations at each and every 1 µm depth section, and visualizing myelinated axons with a lateral resolution of 0.45 µm and an axial resolution of 2 µm.

Modeling of iterative time-reversed ultrasonically encoded optical focusing in a reflection mode.

Time-reversed ultrasonically-encoded (TRUE) optical focusing is a promising technique to realize deep-tissue optical focusing by employing ultrasonic guide stars. However, the sizes of the ultrasound-induced optical focus are determined by the wavelengths of the ultrasound, which are typically tens of microns. To satisfy the need for high-resolution imaging and manipulation, iterative TRUE (iTRUE) was proposed to break this limit by triggering repeated interactions between light and ultrasound and compressing the optical focus. However, even for the best result reported to date, the resolutions along the ultrasound axial and lateral direction were merely improved by only 2-fold to 3-fold. This observation leads to doubt whether iTRUE can be effective in reducing the size of the optical focus. In this work, we address this issue by developing a physical model to investigate iTRUE in a reflection mode numerically. Our numerical results show that, under the influence of shot noises, iTRUE can reduce the optical focus to a single speckle within a finite number of iterations. This model also allows numerical investigations of iTRUE in detail. Quantitatively, based on the parameters set, we show that the optical focus can be reduced to a size of 1.6 µm and a peak-to-background ratio over 104 can be realized. It is also shown that iTRUE cannot significantly advance the focusing depth. We anticipate that this work can serve as useful guidance for optimizing iTRUE system for future biomedical applications, including deep-tissue optical imaging, laser surgery, and optogenetics.

Rational design of pyrrole derivatives with aggregation-induced phosphorescence characteristics for time-resolved and two-photon luminescence imaging

Pure organic room-temperature phosphorescent (RTP) materials have been suggested to be promising bioimaging materials due to their good biocompatibility and long emission lifetime. Herein, we report a class of RTP materials. These materials are developed through the simple introduction of an aromatic carbonyl to a tetraphenylpyrrole molecule and also exhibit aggregation-induced emission (AIE) properties. These molecules show non-emission in solution and purely phosphorescent emission in the aggregated state, which are desirable properties for biological imaging. Highly crystalline nanoparticles can be easily fabricated with a long emission lifetime (20 μs), which eliminate background fluorescence interference from cells and tissues. The prepared nanoparticles demonstrate two-photon absorption characteristics and can be excited by near infrared (NIR) light, making them promising materials for deep-tissue optical imaging. This integrated aggregation-induced phosphorescence (AIP) strategy diversifies the existing pool of bioimaging agents to inspire the development of bioprobes in the future.

Characterization of the spectral memory effect of scattering media.

The optical memory effect is an interesting phenomenon exploited for deep-tissue optical imaging. Besides the widely studied memory effects in the spatial domain to accelerate point scanning speed, the spectral memory effect is also important in multispectral wavefront shaping. Although being theoretically analyzed for decades, quantitative studies of spectral memory effect on a variety of scattering media including biological tissue were rarely reported. In practice, quantifying the range of the spectral memory effect is essential in efficiently shaping broadband light, as it determines the optimum spectral resolution in realizing spatiotemporal focus through scattering media. In this work, we analyze the spectral memory effect based on a diffusion model. An explicit analytical expression involves the illumination wavelength, the diffusion constant, and the sample thickness is derived, which is consistent with the one in the literature. We experimentally quantified the range of spectral correlation for two types of biological tissue, tissue-mimicking phantoms with different concentrations, and diffusers. Specifically, for tissue-mimicking phantoms with calibrated scattering parameters, we show that a correction factor of more than 20 should be inserted, indicating that the range of spectral correlation is much larger than one would expect. This finding is particularly beneficial to multispectral wavefront shaping, as stringent requirements on the spectral resolution could be alleviated by at least one order of magnitude.

Contrast agents for x-ray luminescence computed tomography.

Imaging probes are an important consideration for any type of contrast agent-based imaging method. X-ray luminescence imaging (XLI) and x-ray luminescence computed tomography (XLCT) are both contrast agent-based imaging methods that employ x-ray excitable scintillating imaging probes that emit light to be measured for optical imaging. In this work, we compared the performance of several select imaging probes, both commercial and self-synthesized, for application in XLI/XLCT imaging. Commercially available cadmium telluride quantum dots (CdTe QDs) and europium-doped gadolinium oxysulfide (GOS:Eu) microphosphor as well as synthesized NaGdF4 nanophosphors doped with either europium or terbium were compared through their x-ray luminescence emission spectra, luminescence intensity, and also by performing XLCT scans using phantoms embedded with each of the imaging probes. Each imaging probe displayed a unique emission spectrum that was ideal for deep-tissue optical imaging. In terms of luminescence intensity, due to the large particle size, GOS:Eu had the brightest emission, followed by NaGdF4:Tb, NaGdF4:Eu, and finally the CdTe QDs. Lastly, XLCT scans showed that each imaging probe could be reconstructed with good shape and location accuracy.

Genetic-algorithm-assisted coherent enhancement absorption in scattering media by exploiting transmission and reflection matrices.

The investigations on coherent enhancement absorption (CEA) inside scattering media are critically important in biophotonics. CEA can deliver light to the targeted position, thus enabling deep-tissue optical imaging by improving signal strength and imaging resolution. In this work, we develop a numerical framework that employs the method of finite-difference time-domain. Both the transmission and reflection matrices of scattering media with open boundaries are constructed, allowing the studies on the eigenvalues and eigenchannels. To realize CEA for scattering media with local absorption, we develop a genetic-algorithm-assisted numerical model. By minimizing the total transmittance and reflectance simultaneously, different realizations of CEA are observed and, without setting internal monitors, can be differentiated with cases of light leaked from sides. By modulating the incident wavefront at only one side of the scattering medium, it is shown that for a 5-μm-diameter absorber buried inside a scattering medium of 15 μm × 12 μm, more than half of the incident light can be delivered and absorbed at the target position. The enhancement in absorption is more than four times higher than that with random input. This value can be even higher for smaller absorption regions. We also quantify the effectiveness of the method and show that it is inversely proportional to the openness of the scattering medium. This result is potentially useful for targeted light delivery inside scattering media with local absorption.

Optical transfer function of time-gated coherent imaging in the presence of a scattering medium.

Optical imaging of objects embedded within scattering media such as biological tissues suffers from the loss of resolving power. In our previous work, we proposed an approach called collective accumulation of single scattering (CASS) microscopy that attenuates this detrimental effect of multiple light scattering by combining the time-gated detection and spatial input-output correlation. In the present work, we perform a rigorous theoretical analysis on the effect of multiple light scattering to the optical transfer function of CASS microscopy. In particular, the spatial frequency-dependent signal to noise ratio (SNR) is derived depending on the intensity ratio of the single- and multiple-scattered waves. This allows us to determine the depth-dependent resolving power. We conducted experiments using a Siemens star-like target having various spatial frequency components and supported the theoretical derived SNR spectra. Our study provides a theoretical framework for understanding the effect of multiple light scattering in high-resolution and deep-tissue optical imaging.

Planar and Twisted Molecular Structure Leads to the High Brightness of Semiconducting Polymer Nanoparticles for NIR-IIa Fluorescence Imaging.

Semiconducting polymer nanoparticles (SPNs) emitting in the second near-infrared window (NIR-II, 1000-1700 nm) are promising materials for deep-tissue optical imaging in mammals, but the brightness is far from satisfactory. Herein, we developed a molecular design strategy to boost the brightness of NIR-II SPNs: structure planarization and twisting. By integration of the strong absorption coefficient inherited from planar π-conjugated units and high solid-state quantum yield (ΦPL) from twisted motifs into one polymer, a rise in brightness was obtained. The resulting pNIR-4 with both twisted and planar structure displayed improved ΦPL and absorption when compared to the planar polymer pNIR-1 and the twisted polymer pNIR-2. Given the emission tail extending into the NIR-IIa region (1300-1400 nm) of the pNIR-4 nanoparticles, NIR-IIa fluorescence imaging of blood vessels with enhanced clarity was observed. Moreover, a pH-responsive poly(β-amino ester) made pNIR-4 specifically accumulate at tumor sites, allowing NIR-IIa fluorescence image-guided cancer precision resection. This study provides a molecular design strategy for developing highly bright fluorophores.

Serial optical coherence microscopy for label-free volumetric histopathology

The observation of histopathology using optical microscope is an essential procedure for examination of tissue biopsies or surgically excised specimens in biological and clinical laboratories. However, slide-based microscopic pathology is not suitable for visualizing the large-scale tissue and native 3D organ structure due to its sampling limitation and shallow imaging depth. Here, we demonstrate serial optical coherence microscopy (SOCM) technique that offers label-free, high-throughput, and large-volume imaging of ex vivo mouse organs. A 3D histopathology of whole mouse brain and kidney including blood vessel structure is reconstructed by deep tissue optical imaging in serial sectioning techniques. Our results demonstrate that SOCM has unique advantages as it can visualize both native 3D structures and quantitative regional volume without introduction of any contrast agents.

Self-Illuminating Agents for Deep-Tissue Optical Imaging.

Optical imaging plays an indispensable role in biology and medicine attributing to its noninvasiveness, high spatiotemporal resolution, and high sensitivity. However, as a conventional optical imaging modality, fluorescence imaging confronts issues of shallow imaging depth due to the need for real-time light excitation which produces tissue autofluorescence. By contrast, self-luminescence imaging eliminates the concurrent light excitation, permitting deeper imaging depth and higher signal-to-background ratio (SBR), which has attracted growing attention. Herein, this review summarizes the progress on the development of near-infrared (NIR) emitting self-luminescence agents in deep-tissue optical imaging with highlighting the design principles including molecular- and nano-engineering approaches. Finally, it discusses current challenges and guidelines to develop more effective self-illuminating agents for biomedical diagnosis and treatment.

Development of a beam propagation method to simulate the point spread function degradation in scattering media.

Scattering is one of the main issues that limit the imaging depth in deep tissue optical imaging. To characterize the role of scattering, we have developed a forward model based on the beam propagation method and established the link between the macroscopic optical properties of the media and the statistical parameters of the phase masks applied to the wavefront. Using this model, we have analyzed the degradation of the point-spread function of the illumination beam in the transition regime from ballistic to diffusive light transport. Our method provides a wave-optic simulation toolkit to analyze the effects of scattering on image quality degradation in scanning microscopy. Our open-source implementation is available at https://github.com/BUNPC/Beam-Propagation-Method.

In vivo molecular imaging for immunotherapy using ultra-bright near-infrared-IIb rare-earth nanoparticles.

The NIR-IIb (1500–1700 nm) window is ideal for deep-tissue optical imaging in mammals, but lacks bright and biocompatible probes. Here, we developed biocompatible cubic-phase (α-phase) erbium-based rare-earth nanoparticles (ErNPs) exhibiting bright downconversion luminescence at ~ 1600 nm for dynamic imaging of cancer immune-therapy in mice. We used ErNPs functionalized with cross-linked hydrophilic polymer layers attached to anti-PD-L1 antibody for molecular imaging of PD-L1 in a mouse model of colon cancer and achieved tumor to normal tissue signal ratios of ~ 40. The long luminescence lifetime of ErNPs (~ 4.6 ms) enabled simultaneous imaging of ErNPs and lead sulfide quantum dots (PbS QDs) emitting in the same ~ 1600 nm window. In vivo NIR-IIb molecular imaging of PD-L1 and CD8 revealed cytotoxic T lymphocytes in the tumor microenvironment in response to immunotherapy, and altered CD8 signals in tumor and spleen due to immune activation. The novel crosslinked functionalization layer facilitated 90% ErNPs excretion within two weeks without detectable toxicity in mice.

Deep-tissue optical imaging of near cellular-sized features

Detection of biological features at the cellular level with sufficient sensitivity in complex tissue remains a major challenge. To appreciate this challenge, this would require finding tens to hundreds of cells (a 0.1 mm tumor has ~125 cells), out of ~37 trillion cells in the human body. Near-infrared optical imaging holds promise for high-resolution, deep-tissue imaging, but is limited by autofluorescence and scattering. To date, the maximum reported depth using second-window near-infrared (NIR-II: 1000–1700 nm) fluorophores is 3.2 cm through tissue. Here, we design an NIR-II imaging system, “Detection of Optically Luminescent Probes using Hyperspectral and diffuse Imaging in Near-infrared” (DOLPHIN), that resolves these challenges. DOLPHIN achieves the following: (i) resolution of probes through up to 8 cm of tissue phantom; (ii) identification of spectral and scattering signatures of tissues without apriori knowledge of background or autofluorescence; and (iii) 3D reconstruction of live whole animals. Notably, we demonstrate noninvasive real-time tracking of a 0.1 mm-sized fluorophore through the gastrointestinal tract of a living mouse, which is beyond the detection limit of current imaging modalities.

Synergy of light and sound for deep-tissue optical imaging and focusing

Light and sound are the two most dominant mechanisms with which we naturally perceive this world. Both have their significant impacts yet with inherent limitations. For example, sound is insensitive to soft tissue functional changes, and light is strongly scattered in tissue, resulting in a trade-off between penetration depth and resolution. Here, we present our most recent research efforts of exploiting the synergy of light and sound to overcome this limitation. Specifically, photoacoustics converts diffusive photon into non-scattering ultrasonic waves, enabling a high-contrast sensing of optical absorption with ultrasonic resolution in deep tissue, overcoming the optical diffusion limit from the signal detection perspective. The generation of photoacoustic signals, however, is still throttled by the attenuation of photon flux due to the strong diffusion effect of light in tissue. Therefore, wavefront shaping is introduced, so that multiply scattered light could be manipulated so as to retain optical focusing or sufficient photon flux even at depths in tissue. We will present the recent development of photoacoustic imaging and optical wavefront shaping in our lab. Potential applications, existing challenges, and further improvement are also discussed.Light and sound are the two most dominant mechanisms with which we naturally perceive this world. Both have their significant impacts yet with inherent limitations. For example, sound is insensitive to soft tissue functional changes, and light is strongly scattered in tissue, resulting in a trade-off between penetration depth and resolution. Here, we present our most recent research efforts of exploiting the synergy of light and sound to overcome this limitation. Specifically, photoacoustics converts diffusive photon into non-scattering ultrasonic waves, enabling a high-contrast sensing of optical absorption with ultrasonic resolution in deep tissue, overcoming the optical diffusion limit from the signal detection perspective. The generation of photoacoustic signals, however, is still throttled by the attenuation of photon flux due to the strong diffusion effect of light in tissue. Therefore, wavefront shaping is introduced, so that multiply scattered light could be manipulated so as to retain optical foc...