Deep Inside Tissue Research Articles

A Water‐Soluble Fluorescent Probe Derived from Pyridine‐2,6‐Dicarboxylic Acid: Synthesis, Interaction with Ions, and Two‐Photon Microscopy

AbstractTwo‐photon excited fluorescence is a process in which the excited fluorophore relaxes and emits fluorescence after two‐photon absorption. Combined with appropriate two‐photon fluorescent probes, two‐photon microscopy enables observation deep inside tissue without damage. Former reports showed that the two‐photon absorption cross sections of donor–acceptor (D–A) dipoles, as well as D‐π‐D and D–A–D quadrupoles can be strengthened by using strong D–A groups. In this study, a novel water‐soluble fluorescent probe with D‐π‐A structure containing pyridine‐2,6‐dicarboxylic acid unit was synthesized. The maximum excitation wavelength and emission wavelength of the probe in aqueous solution were 328 nm and 471 nm, respectively. The fluorescence emitted by the probe can be quenched by various metal cations. The pyridyl N and carboxyl O in the probe formed chelates with Cu2+, which resulted in a fluorescence quenching response, and showed a linear relationship between fluorescent intensity and Cu2+ concentration with wide linear range and low limit of detection. CCK‐8 tests showed the probe had no obvious toxicity to 4T1 cells in the range of 0.4–50 μmol L−1, and the cell survival rate was above 86 %. The probe had a large two‐photon absorption cross section ranging from 680 nm to 820 nm. The two‐photon absorption cross section reached 100 GM at 760 nm of excitation wavelength. By means of microscopic imaging of 4T1 cells stained with the probe, one‐photon fluorescence represented blue emission, while two‐photon fluorescence represented bright green emission. The probe was proved to have good cell permeability, and preferred to target nucleus, showing application potential in two‐photon fluorescence imaging technology.

Full-Vectorial Light Propagation Simulation of Optimized Beams in Scattering Media

Volumetric scattering prevents imaging modalities in biomedical optics from imaging deep inside tissue. The optimization of the incident wavefront has the potential to improve these imaging modalities. To investigate the optimization and light propagation of such beams inside scattering media rigorously, full-vectorial simulations based on solutions of Maxwell’s equations are necessary. In this publication, we present a versatile two-step beam synthesis method to efficiently simulate the scanning and phase optimization of a focused beam inside a static scattering medium. We present four different approaches to the phase optimization of the energy density and the absolute value of the Poynting vector. We find that these quantities have two regions with different, almost exponential decays over depth for a non-optimized beam. Optimization by conjugating the phase of the projected electric field in various directions at the focus shows an improvement below a certain penetration depth. Seeking global solutions to the optimization problems reveals an even better enhancement in the energy density and the absolute value of the Poynting vector in the focus. For Poynting vector optimization, the differences between the presented optimization approaches are more significant than for the energy density. With the presented method, it is possible to efficiently simulate different imaging methods improved by wavefront shaping to investigate their possible penetration depths.

Optoacoustic Endoscopy of the Gastrointestinal Tract

Optical endoscopy is the most commonly applied procedure for inspecting the gastrointestinal (GI) tract, and it is based on different approaches and designs, from using a flexible optical scope to swallowing a small camera capsule that obtains photographs as it advances through the digestive tract. Despite its wide use in GI diagnostics and theranostics, optical visualization only allows a superficial inspection of the wall lining (mucosa), therefore limiting the ability to obtain information from deeper GI layers. In the quest for developing methods to visualize under the mucosal layers, we review herein progress with optoacoustic endoscopy, a technique that captures optical contrast in high resolution deep inside tissues, enabling imaging beneath the surface of the mucosa. Optoacoustic endoscopy combines imaging of optical contrast with the resolution and depth penetration afforded by ultrasonography, thus merging highly advantageous characteristics for clinical applications. We review progress and the current status of the technology, its key endoscopic competitors, and challenges for clinical application. We further offer a perspective regarding future directions and the overall application potential of the technique to complement the current state-of-the-art.

Proton-Only Sensing of Hyperpolarized [1,2-13C2]Pyruvate.

Hyperpolarized MRI is emerging as a next-generation molecular imaging modality that can detect metabolic transformations in real time deep inside tissue and organs. 13C-hyperpolarized pyruvate is the leading hyperpolarized contrast agent that can probe cellular energetics in real time. Currently, hyperpolarized MRI requires specialized "multinuclear" MRI scanners that have the ability to excite and detect 13C signals. The objective of this work is the development of an approach that works on conventional (i.e., proton-only) MRI systems while taking advantage of long-lived 13C hyperpolarization. The long-lived singlet state of [1,2-13C2]pyruvate is hyperpolarized with parahydrogen in reversible exchange, and subsequently, the polarization is transferred from the 13C2 spin pair to the methyl protons of pyruvate for detection. This polarization transfer is accomplished with spin-lock induced crossing pulses that are only applied to the methyl protons yet access the hyperpolarization stored in the 13C2 singlet state. Theory and first experimental demonstrations are provided for our method, which obviates 13C excitation and detection for proton sensing of 13C-hyperpolarized pyruvate with an overall experimental-polarization transfer efficiency of ∼22% versus a theoretically predicted polarization transfer efficiency of 25%.

Computational microscopy for fast widefield deep-tissue fluorescence imaging using a commercial dual-cannula probe.

A solid-glass cannula serves as a micro-endoscope that can deliver excitation light deep inside tissue while also collecting emitted fluorescence. Then, we utilize deep neural networks to reconstruct images from the collected intensity distributions. By using a commercially available dual-cannula probe, and training a separate deep neural network for each cannula, we effectively double the field of view compared to prior work. We demonstrated ex vivo imaging of fluorescent beads and brain slices and in vivo imaging from whole brains. We clearly resolved 4 μm beads, with FOV from each cannula of 0.2 mm (diameter), and produced images from a depth of ~1.2 mm in the whole brain, currently limited primarily by the labeling. Since no scanning is required, fast widefield fluorescence imaging limited primarily by the brightness of the fluorophores, collection efficiency of our system, and the frame rate of the camera becomes possible.

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 Cost-Efficient Multiwavelength LED-Based System for Quantitative Photoacoustic Measurements.

The unique ability of photoacoustic (PA) sensing to provide optical absorption information of biomolecules deep inside turbid tissues with high sensitivity has recently enabled the development of various novel diagnostic systems for biomedical applications. In many cases, PA setups can be bulky, complex, and costly, as they typically require the integration of expensive Q-switched nanosecond lasers, and also presents limited wavelength availability. This article presents a compact, cost-efficient, multiwavelength PA sensing system for quantitative measurements, by utilizing two high-power LED sources emitting at central wavelengths of 444 and 628 nm, respectively, and a single-element ultrasonic transducer at 3.5 MHz for signal detection. We investigate the performance of LEDs in pulsed mode and explore the dependence of PA responses on absorber’s concentration and applied energy fluence using tissue-mimicking phantoms demonstrating both optical absorption and scattering properties. Finally, we apply the developed system on the spectral unmixing of two absorbers contained at various relative concentrations in the phantoms, to provide accurate estimations with absolute deviations ranging between 0.4 and 12.3%. An upgraded version of the PA system may provide valuable in-vivo multiparametric measurements of important biomarkers, such as hemoglobin oxygenation, melanin concentration, local lipid content, and glucose levels.

Single-shot ultrasound-modulated optical tomography with enhanced speckle contrast.

Ultrasound-modulated optical tomography (UOT) images optical contrast deep inside biological tissue. Among existing approaches, camera-based parallel detection is beneficial in modulation depth but is limited to the relatively slow framerate of cameras. This condition prevents such a scheme from achieving maturity to image live animals with sub-millisecond speckle correlation time. In this work, we developed on-axis single-shot UOT by investigating the statistics of speckles, breaking the restriction imposed by the slow camera framerate. As a proof of concept, we experimentally imaged a one-dimensional absorptive object buried inside a moving scattering medium with speckle correlation time down to 0.48 ms. We envision that this single-shot UOT is promising to cope with live animals with fast speckle decorrelation.

Biocompatible diameter-oscillating fiber with microlens endface.

Optical fibers have been widely applied to life science, such as laser delivering, fluorescence collection, biosensing, bioimaging, etc. To resolve the challenges of advanced multiphoton biophotonic applications utilizing ultrashort laser pulses, here we report a flexible diameter-oscillating fiber (DOF) with microlens endface fabricated by using Polydimethylsiloxane (PDMS) elastomers. The diameter of the DOF is designed to longitudinally vary for providing accurate dispersion management, which is important for near-infrared multiphoton biophotonics that usually involves ultrashort laser pulses. The variation range and period of the DOF's diameter can be flexibly adjusted by controlling the parameters during the fabrication, such that dispersion curves with different oscillation landscapes can be obtained. The dispersion oscillating around the zero-dispersion baseline gives rise to a minimized net dispersion as the ultrashort laser pulse passes through the DOF - reducing the temporal broadening effect and resulting in transform-limited pulsewidth. In addition, the endface of the DOF is fabricated with a microlens, which is especially useful for laser scanning/focusing and fluorescence excitation. It is anticipated that this new biocompatible DOF is of great interest for biophotonic applications, particularly multiphoton microscopy deep inside biological tissues.

3D super-resolution deep-tissue imaging in living mice.

Stimulated emission depletion (STED) microscopy enables the three-dimensional (3D) visualization of dynamic nanoscale structures in living cells, offering unique insights into their organization. However, 3D-STED imaging deep inside biological tissue is obstructed by optical aberrations and light scattering. We present a STED system that overcomes these challenges. Through the combination of two-photon excitation, adaptive optics, red-emitting organic dyes, and a long-working-distance water-immersion objective lens, our system achieves aberration-corrected 3D super-resolution imaging, which we demonstrate 164 µm deep in fixed mouse brain tissue and 76 µm deep in the brain of a living mouse.

Distinction of surgically resected gastrointestinal stromal tumor by near-infrared hyperspectral imaging

The diagnosis of gastrointestinal stromal tumor (GIST) using conventional endoscopy is difficult because submucosal tumor (SMT) lesions like GIST are covered by a mucosal layer. Near-infrared hyperspectral imaging (NIR-HSI) can obtain optical information from deep inside tissues. However, far less progress has been made in the development of techniques for distinguishing deep lesions like GIST. This study aimed to investigate whether NIR-HSI is suitable for distinguishing deep SMT lesions. In this study, 12 gastric GIST lesions were surgically resected and imaged with an NIR hyperspectral camera from the aspect of the mucosal surface. Thus, the images were obtained ex-vivo. The site of the GIST was defined by a pathologist using the NIR image to prepare training data for normal and GIST regions. A machine learning algorithm, support vector machine, was then used to predict normal and GIST regions. Results were displayed using color-coded regions. Although 7 specimens had a mucosal layer (thickness 0.4–2.5 mm) covering the GIST lesion, NIR-HSI analysis by machine learning showed normal and GIST regions as color-coded areas. The specificity, sensitivity, and accuracy of the results were 73.0%, 91.3%, and 86.1%, respectively. The study suggests that NIR-HSI analysis may potentially help distinguish deep lesions.

A Deep Learning Approach to Photoacoustic Wavefront Localization in Deep-Tissue Medium.

Optical photons undergo strong scattering when propagating beyond 1-mm deep inside biological tissue. Finding the origin of these diffused optical wavefronts is a challenging task. Breaking through the optical diffusion limit, photoacoustic (PA) imaging (PAI) provides high-resolution and label-free images of human vasculature with high contrast due to the optical absorption of hemoglobin. In real-time PAI, an ultrasound transducer array detects PA signals, and B-mode images are formed by delay-and-sum or frequency-domain beamforming. Fundamentally, the strength of a PA signal is proportional to the local optical fluence, which decreases with the increasing depth due to depth-dependent optical attenuation. This limits the visibility of deep-tissue vasculature or other light-absorbing PA targets. To address this practical challenge, an encoder-decoder convolutional neural network architecture was constructed with custom modules and trained to identify the origin of the PA wavefronts inside an optically scattering deep-tissue medium. A comprehensive ablation study provides strong evidence that each module improves the localization accuracy. The network was trained on model-based simulated PA signals produced by 16 240 blood-vessel targets subjected to both optical scattering and Gaussian noise. Test results on 4600 simulated and five experimental PA signals collected under various scattering conditions show that the network can localize the targets with a mean error less than 30 microns (standard deviation 20.9 microns) for targets below 40-mm imaging depth and 1.06 mm (standard deviation 2.68 mm) for targets at a depth between 40 and 60 mm. The proposed work has broad applications such as diffused optical wavefront shaping, circulating melanoma cell detection, and real-time vascular surgeries (e.g., deep-vein thrombosis).

3D computational cannula fluorescence microscopy enabled by artificial neural networks.

Computational cannula microscopy (CCM) is a high-resolution widefield fluorescence imaging approach deep inside tissue, which is minimally invasive. Rather than using conventional lenses, a surgical cannula acts as a lightpipe for both excitation and fluorescence emission, where computational methods are used for image visualization. Here, we enhance CCM with artificial neural networks to enable 3D imaging of cultured neurons and fluorescent beads, the latter inside a volumetric phantom. We experimentally demonstrate transverse resolution of ∼6µm, field of view ∼200µm and axial sectioning of ∼50µm for depths down to ∼700µm, all achieved with computation time of ∼3ms/frame on a desktop computer.

Parallelized Monte Carlo Photon Transport Simulations for Arbitrary Multi-Angle Wide-Field Illumination in Optoacoustic Imaging

Optoacoustic imaging (OAI) or photoacoustic imaging can resolve the distribution of tissue chromophores and optical contrast agents deep inside tissue from the optoacoustic detection with multi-spectral illumination. The development of a fast and accurate modeling method for the photon transport in OAI is necessary to quantitatively evaluate the tissue optical parameters. This paper presents a parallelized Monte Carlo modeling method especially for OAI (MCOAI) to simulate photon transport in bio-tissues with arbitrary multi-angle wide-field illumination. The performance of the MCOAI method is verified by comparison with the graphics processing unit (GPU)-accelerated MCX method in the typical cases with the pencil beam and ring source illumination. The simulation results demonstrate the GPU-based MCOAI method has equivalent accuracy and significantly improved computation efficiency, compared to the MCX method. The simulations with cylindrical and hemispherical source illumination further illustrate that the MCOAI method can effectively implement three-dimensional photon transport simulation for typical illumination geometries of OAI systems. A cross-section of Digimouse is selected as a realistic heterogeneous phantom illuminated with six different light sources usually employed in OAI, in order to prove the necessity of establishing photon transport modeling in OAI for the quantitative visualization in deep tissues. The MCOAI method can provide a powerful tool to efficiently establish photon transport modeling with arbitrary illumination modes for OAI applications.

Molecular Fluorophores for Deep-Tissue Bioimaging.

Fluorescence imaging has made tremendous inroads toward understanding the complexity of biological systems, but in vivo deep-tissue imaging remains a great challenge due to the optical opacity of biological tissue. Recent improvements in laser and detector manufacturing have allowed the expansion of nonlinear and linear fluorescence imaging to the underexplored “tissue-transparent” second near-infrared (NIR-II; 1000–1700 nm) window, opening up new opportunities for optical access deep inside opaque tissue. Molecular fluorophores have historically played a major role in fluorescence bioimaging. It is increasingly important to design new molecular fluorophores to fully unlock the potential of NIR-II imaging techniques. In this outlook, we give an overview of the novel molecular fluorophores developed for deep-tissue bioimaging in the past five years and discuss their pros and cons in applications. Guidelines for designing new molecular fluorophores with the desirable properties are also provided.

Self-Reporting and Splitting Nanopomegranates Potentiate Deep Tissue Cancer Radiotherapy via Elevated Diffusion and Transcytosis.

The efficacy of nanoradiosensitizers in cancer therapy has been primarily impeded by their limited accessibility to radioresistant cancer cells residing deep inside tumor tissues. The failure to report tumor response to radiotherapy generally delays adjustment of the treatment schedule and sets up another substantial obstacle to clinical success. Here, we develop a nanopomegranate (RNP) platform that not only visualizes the cancer radiosensitivities but also potentiates deep tissue cancer radiotherapy via elevated passive diffusion and active transcytosis. The RNPs are engineered through the programmed self-assembly of a tumor environment-targeting polymeric matrix and modular building blocks of ultrasmall gold nanoparticles (Au5). Once RNPs reach the tumors, the environmental acidity triggers the splitting and surface cationization of Au5. The small dimension of Au5 allows its passive diffusion, while positive surface charge enables its active transcytosis to cross the tumor interstitium. Meanwhile, the reporter element monitors the feedback of favorable radiotherapy responsiveness by detecting the activated apoptosis after radiation. The pivotal role of RNPs in improving and identifying radiotherapeutic outcomes is demonstrated in various tumor bearing mouse models with different radiosensitivities. In summary, our strategy offers a promising paradigm for deep tissue drug delivery as well as individualized precision radiotherapy.

Three-dimensional nanoscopy of whole cells and tissues with in situ point spread function retrieval.

Single-molecule localization microscopy is a powerful tool for visualizing subcellular structures, interactions, and protein functions in biological research. However, inhomogeneous refractive indices inside cells and tissues distort the fluorescent signal emitted from single-molecule probes, which rapidly deteriorates resolution with increasing depth. We propose a method that enables the construction of an in situ 3D response of single emitters directly from single-molecule blinking datasets and therefore allows their locations to be pin-pointed with precision that achieves the Cramer-Rao lower bound and uncompromised fidelity. We demonstrate this method, named in situ PSF retrieval (INSPR), across a range of cellular and tissue architectures from mitochondrial networks and nuclear pores in mammalian cells, to amyloid β plaques and dendrites in brain tissues, and elastic fibers in developing cartilage of mice. This advancement expands the routine applicability of super-resolution microscopy from selected cellular targets near coverslips to intra- and extra-cellular targets deep inside tissues.

Analytical modeling of depth-dose degradation in heterogeneous lung tissue for intensity-modulated proton therapy planning.

Proton therapy may be promising for treating non-small-cell lung cancer due to lower doses to the lung and heart, as compared to photon therapy. A reported challenge is degradation, i.e., a smoothing of the depth-dose distribution due to heterogeneous lung tissue. For pencil beams, this causes a distal falloff widening and a peak-to-plateau ratio decrease, not considered in clinical treatment planning systems. We present a degradation model implemented into an analytical dose calculation, fully integrated into a treatment planning workflow. Degradation effects were investigated on target dose, distal dose falloffs, and mean lung dose for ten patient cases with varying anatomical characteristics. For patients with pronounced range straggling (in our study large tumors, or lesions close to the mediastinum), degradation effects were restricted to a maximum decrease in target coverage (D 95 of the planning target volume) of 1.4%. The median broadening of the distal 80-20% dose falloffs was 0.5mm at the maximum. For small target volumes deep inside lung tissue, however, the target underdose increased considerably by up to 26%. The mean lung dose was not negatively affected by degradation in any of the investigated cases. For most cases, dose degradation due to heterogeneous lung tissue did not yield critical organ at risk overdosing or overall target underdosing. However, for small and deep-seated tumors which can only be reached by penetrating lung tissue, we have seen substantial local underdose, which deserves further investigation, also considering other prevalent sources of uncertainty.

Computational cannula microscopy of neurons using neural networks.

Computational cannula microscopy is a minimally invasive imaging technique that can enable high-resolution imaging deep inside tissue. Here, we apply artificial neural networks to enable real-time, power-efficient image reconstructions that are more efficiently scalable to larger fields of view. Specifically, we demonstrate widefield fluorescence microscopy of cultured neurons and fluorescent beads with a field of view of 200 µm (diameter) and a resolution of less than 10 µm using a cannula of diameter of only 220 µm. In addition, we show that this approach can also be extended to macro-photography.

Light-Emitting-Diode-Based Multispectral Photoacoustic Computed Tomography System.

Photoacoustic computed tomography (PACT) has been widely explored for non-ionizing functional and molecular imaging of humans and small animals. In order for light to penetrate deep inside tissue, a bulky and high-cost tunable laser is typically used. Light-emitting diodes (LEDs) have recently emerged as cost-effective and portable alternative illumination sources for photoacoustic imaging. In this study, we have developed a portable, low-cost, five-dimensional (x, y, z, t, PACT system using multi-wavelength LED excitation to enable similar functional and molecular imaging capabilities as standard tunable lasers. Four LED arrays and a linear ultrasound transducer detector array are housed in a hollow cylindrical geometry that rotates 360 degrees to allow multiple projections through the subject of interest placed inside the cylinder. The structural, functional, and molecular imaging capabilities of the LED–PACT system are validated using various tissue-mimicking phantom studies. The axial, lateral, and elevational resolutions of the system at 2.3 cm depth are estimated as 0.12 mm, 0.3 mm, and 2.1 mm, respectively. Spectrally unmixed photoacoustic contrasts from tubes filled with oxy- and deoxy-hemoglobin, indocyanine green, methylene blue, and melanin molecules demonstrate the multispectral molecular imaging capabilities of the system. Human-finger-mimicking phantoms made of a bone and blood tubes show structural and functional oxygen saturation imaging capabilities. Together, these results demonstrate the potential of the proposed LED-based, low-cost, portable PACT system for pre-clinical and clinical applications.