Deep-tissue Optical Imaging Research Articles

Development of organic semiconducting materials for deep-tissue optical imaging, phototherapy and photoactivation.

Biophotonics as a highly interdisciplinary frontier often requires the assistance of optical agents to control the light pathways in cells, tissues and living organisms for specific biomedical applications. Organic semiconducting materials (OSMs) composed of π-conjugated building blocks as the optically active components have recently emerged as a promising category of biophotonic agents. OSMs possess common features including excellent optical properties, good photostability and biologically benign composition. This review summarizes the recent progress in the development of OSMs based on small-molecule fluorophores, aggregation-induced emission (AIE) dyes and semiconducting oligomer/polymer nanoparticles (SONs/SPNs) for advanced biophotonic applications. OSMs have been exploited as imaging agents to transduce biomolecular interactions into second near-infrared fluorescence, chemiluminescence, afterglow or photoacoustic signals, enabling deep-tissue ultrasensitive imaging of biological tissues, disease biomarkers and physiological indexes. By fine-tuning the molecular structures, OSMs can also convert light energy into cytotoxic free radicals or heat, allowing for effective cancer phototherapy. Due to their instant light response and efficient light-harvesting properties, precise regulation of biological activities using OSMs as remote transducers has been demonstrated for protein ion channels, gene transcription and protein activation. In addition to highlighting OSMs as a multifunctional platform for a wide range of biomedical applications, current challenges and perspectives of OSMs in biophotonics are discussed.

Switchable Photoacoustic Imaging of Glutathione Using MnO2 Nanotubes for Cancer Diagnosis.

Glutathione is overexpressed in tumor cells and regulates cancer growth, metastasis, and drug resistance. Therefore, detecting glutathione levels may greatly facilitate cancer diagnosis and treatment response monitoring. Photoacoustic (PA) imaging is a noninvasive modality for high-sensitivity, high-resolution, deep-tissue optical imaging. Switchable PA probes that offer signal on/off responses to tumor targets would further improve the detection sensitivity and signal-to-noise ratio of PA imaging. Here, we explore the use of MnO2 nanotubes as a switchable and biodegradable PA probe for dynamic imaging of glutathione in cancer. Glutathione reduces black MnO2 nanotubes into colorless Mn2+ ions, leading to decreased and signal off PA amplitude. In phantoms, we observed a linear response of reduced PA signals of MnO2 nanotubes to increased glutathione concentrations. Using melanoma as the disease model, we demonstrated that MnO2 nanotube-based PA imaging of glutathione successfully distinguished B16F10 melanoma cells from BEAS-2B normal cells and discriminated B16F10 tumors from healthy skin tissues. Our results showed that MnO2 nanotubes are a potent switchable and biodegradable PA probe for glutathione imaging in cancer diagnosis.

Upconverting nanoparticle micro-lightbulbs designed for deep tissue optical stimulation and imaging.

Optical methods for imaging and stimulation of biological events based on the use of visible light are limited to the superficial layers of tissue due to the significant absorption and scattering of light. Here, we demonstrate the design and implementation of passive micro-structured lightbulbs (MLBs) containing bright-emitting lanthanide-doped upconverting nanoparticles (UCNPs) for light delivery deep into the tissue. The MLBs are realized as cylindrical pillars made of Parylene C polymer that can be implanted deep into the tissue. The encapsulated UCNPs absorb near-infrared (NIR) light at λ = 980 nm, which undergoes much less absorption than the blue light in the brain tissue, and then locally emit blue light (1G4→3H6 and 1D2→3F4 transitions) that can be used for optogenetic excitation of neurons in the brain. The 3H4→3H6 transition will result in the emission of higher energy NIR photons at λ = 800 nm that can be used for imaging and tracking MLBs through thick tissue.

Organic Semiconducting Agents for Deep-Tissue Molecular Imaging: Second Near-Infrared Fluorescence, Self-Luminescence, and Photoacoustics.

Optical imaging has played a pivotal role in biology and medicine, but it faces challenges of relatively low tissue penetration and poor signal-to-background ratio due to light scattering and tissue autofluorescence. To overcome these issues, second near-infrared fluorescence, self-luminescence, and photoacoustic imaging have recently emerged, which utilize an optical region with reduced light-tissue interactions, eliminate real-time light excitation, and detect acoustic signals with negligible attenuation, respectively. Because there are only a few endogenous molecules absorbing or emitting above the visible region, development of contrast agents is essential for those deep-tissue optical imaging modalities. Organic semiconducting agents with π-conjugated frameworks can be synthesized to meet different optical imaging requirements due to their easy chemical modification and legible structure-property relation. Herein, the deep-tissue optical imaging applications of organic semiconducting agents including small-molecule agents and nanoparticle derivatives are summarized. In particular, the molecular engineering and nanoformulation approaches to further improve the tissue penetration and detection sensitivity of these optical imaging modalities are highlighted. Finally, current challenges and potential opportunities in this emerging subfield of biomedical imaging are discussed.

Lanthanide-Doped Core-Shell-Shell Nanocomposite for Dual Photodynamic Therapy and Luminescence Imaging by a Single X-ray Excitation Source.

Photodynamic therapy (PDT) could be highly selective and noninvasive, with low side effects as an adjuvant therapy for cancer treatment. Because excitation sources such as UV and visible lights for most of the photosensitizers do not penetrate deeply enough into biological tissues, PDT is useful only when the lesions are located within 10 mm below the skin. In addition, there is no prior example of theranostics capable of both PDT and imaging with a single deep-penetrating X-ray excitation source. Here we report a new theranostic scintillator nanoparticle (ScNP) composite in a core-shell-shell arrangement, that is, NaLuF4:Gd(35%),Eu(15%)@NaLuF4:Gd(40%)@NaLuF4:Gd(35%),Tb(15%), which is capable of being excited by a single X-ray radiation source to allow potentially deep tissue PDT and optical imaging with a low dark cytotoxicity and effective photocytotoxicity. With the X-ray excitation, the ScNPs can emit visible light at 543 nm (from Tb3+) to stimulate the loaded rose bengal (RB) photosensitizer and cause death of efficient MDA-MB-231 and MCF-7 cancer cells. The ScNPs can also emit light at 614 and 695 nm (from Eu3+) for luminescence imaging. The middle shell in the core-shell-shell ScNPs is unique to separate the Eu3+ in the core and the Tb3+ in the outer shell to prevent resonance quenching between them and to result in good PDT efficiency. Also, it was demonstrated that although the addition of a mesoporous SiO2 layer resulted in the transfer of 82.7% fluorescence resonance energy between Tb3+ and RB, the subsequent conversion of the energy from RB to generate 1O2 was hampered, although the loaded amount of the RB was almost twice that without the mSiO2 layer. A unique method to compare the wt % and mol % compositions calculated by using the morphological transmission electron microscope images and the inductively coupled plasma elemental analysis data of the core, core-shell, and core-shell-shell ScNPs is also introduced.

Super-resolution photoacoustic imaging via flow-induced absorption fluctuations

In deep-tissue photoacoustic imaging, optical-contrast images of deep-lying structures are formed by recording acoustic waves that are generated by optical absorption. Although photoacoustics is perhaps the leading technique for high-resolution deep-tissue optical imaging, its spatial resolution is fundamentally limited by the acoustic wavelength, which is orders of magnitude longer than the optical diffraction limit. Here, we present an approach for surpassing the acoustic diffraction limit in photoacoustics by exploiting inherent temporal fluctuations in the photoacoustic signals due to sample dynamics, such as those induced by the flow of absorbing red blood cells. This was achieved using a conventional photoacoustic imaging system by adapting concepts from super-resolution fluorescence fluctuation microscopy to the statistical analysis of acoustic signals from flowing acoustic emitters. Specifically, we experimentally demonstrate that flow of absorbing particles and whole human blood yields super-resolved photoacoustic images, and provides static background reduction. By generalizing the statistical analysis to complex-valued signals, we demonstrate super-resolved photoacoustic images that are free from common photoacoustic imaging artifacts caused by band-limited acoustic detection. The presented technique holds potential for contrast-agent-free microvessel imaging, as red blood cells provide a strong endogenous source of naturally fluctuating absorption.

Bright X-ray and up-conversion nanophosphors annealed using encapsulated sintering agents for bioimaging applications.

Nanophosphors are promising contrast agents for deep tissue optical imaging applications because they can be excited by X-ray and near infrared light that penetrates deeply through tissue and generates almost no autofluorescence background in the tissue. For these bioimaging applications, the nanophosophors should ideally be small, monodispersed and brightly luminescent. However, most methods used to improve luminescence yield by annealing the particles to reduce crystal and surface defects (e.g. using flux or sintering agents) also cause particle fusion or require multiple component core-shell structures. Here, we report a novel method to prepare bright, uniformly sized X-ray nanophosphors (Gd2O2S:Eu or Tb) and upconversion nanophosphors (Y2O2S: Yb/Er, or Yb/Tm) with large crystal domain size without causing aggregation. A core-shell nanoparticle is formed, with NaF only in the core. We observe that increasing the NaF sintering agent concentration up to 7.6 mol% increases both crystal domain size and luminescence intensity (up to 40% of commercial microphosphors) without affecting the physical particticle diameter. Above 7.6 mol%, particle fusion is observed. The annealing is insensitive to the cation (Na+ or K+) but varies strongly with anion, with F->Cl->CO32->Br->I-. The luminescence depends strongly on crystal domain size. The data agree reasonably well with a simple domain surface quenching model, although the size-dependence suggests additional quenching mechanisms within small domains. The prepared bright nanophosphors were subsequently functionalized with PEG-folic acid to target MCF-7 breast cancer cells which overexpress folic acid receptors. Both X-ray and upconversion nanophosphors provided low background and bright luminescence which was imaged through 1 cm chicken breast tissue at a low dose of nanophosphors 200 µL (0.1 mg/mL). We anticipate these highly monodispersed and bright X-ray and upconversion nanophosphors will have significant potential for tumor targeted imaging.

Focusing light through biological tissue and tissue-mimicking phantoms up to 9.6cm in thickness with digital optical phase conjugation.

Optical phase conjugation (OPC)-based wavefront shaping techniques focus light through or within scattering media, which is critically important for deep-tissue optical imaging, manipulation, and therapy. However, to date, the sample thickness in OPC experiments has been limited to only a few millimeters. Here, by using a laser with a long coherence length and an optimized digital OPC system that can safely deliver more light power, we focused 532-nm light through tissue-mimicking phantoms up to 9.6 cm thick, as well as through ex vivo chicken breast tissue up to 2.5 cm thick. Our results demonstrate that OPC can be achieved even when photons have experienced on average 1000 scattering events. The demonstrated penetration of nearly 10 cm (∼100 transport mean free paths) has never been achieved before by any optical focusing technique, and it shows the promise of OPC for deep-tissue noninvasive optical imaging, manipulation, and therapy.

Real time monitoring of aminothiol level in blood using a near-infrared dye assisted deep tissue fluorescence and photoacoustic bimodal imaging.

The development of molecular probes for the detection and imaging of biological thiols is a major step forward diagnosing various types of diseases. Previously reported thiol imaging strategies were mainly based on a single mode of imaging with a limited application in vivo. In this work, we introduced an unsymmetrical near-infrared (NIR) squaraine dye (USq) as an exogenous contrast agent for photoacoustic and fluorescence bimodal imaging of thiol variations in live animals. USq exhibits a narrow absorption band at 680 nm that generates a photoacoustic signal and a strong NIR emission at 700 nm (ΦF = 0.27), which is applicable for deep tissue optical imaging. Both photoacoustic and fluorescence signals could selectively disappear in the presence of different thiols. Through in vitro and in vivo imaging studies, unique imaging capability of USq was demonstrated, and the effect of food uptake on the increased level of aminothiols in blood was confirmed.

Single-exposure optical focusing inside scattering media using binarized time-reversed adapted perturbation.

Light scattering inhibits high-resolution optical imaging, manipulation and therapy deep inside biological tissue by preventing focusing. To form deep foci, wavefront shaping techniques that break the optical diffusion limit have been developed. For in vivo applications, such focusing must provide high gain, high speed, and a high focal peak-to-background ratio. However, none of the previous techniques meet these requirements simultaneously. Here, we overcome this challenge by rapidly measuring the perturbed optical field within a single camera exposure followed by adaptively time-reversing the phase-binarized perturbation. Consequently, a phase-conjugated wavefront is synthesized within a millisecond, two orders of magnitude shorter than the digitally achieved record. We demonstrated real-time focusing in dynamic scattering media, and extended laser speckle contrast imaging to new depths. The unprecedented combination of fast response, high gain, and high focusing contrast makes this work a major stride toward in vivo deep tissue optical imaging, manipulation, and therapy.

Exploring anti-reflection modes in disordered media.

Sensing and manipulating targets hidden under scattering media are universal problems that take place in applications ranging from deep-tissue optical imaging to laser surgery. A major issue in these applications is the shallow light penetration caused by multiple scattering that reflects most of incident light. Although advances have been made to eliminate image distortion by a scattering medium, dealing with the light reflection has remained unchallenged. Here we present a method to minimize reflected intensity by finding and coupling light into the anti-reflection modes of a scattering medium. In doing so, we achieved more than a factor of 3 increase in light penetration. Our method of controlling reflected waves makes it readily applicable to in vivo applications in which detector sensors can only be positioned at the same side of illumination and will therefore lay the foundation of advancing the working depth of many existing optical imaging and treatment technologies.

Relation between speckle decorrelation and optical phase conjugation (OPC)-based turbidity suppression through dynamic scattering media: a study on in vivo mouse skin.

Light scattering in biological tissue significantly limits the accessible depth for localized optical interrogation and deep-tissue optical imaging. This challenge can be overcome by exploiting the time-reversal property of optical phase conjugation (OPC) to reverse multiple scattering events or suppress turbidity. However, in living tissue, scatterers are highly movable and the movement can disrupt time-reversal symmetry when there is a latency in the OPC playback. In this paper, we show that the motion-induced degradation of the OPC turbidity-suppression effect through a dynamic scattering medium shares the same decorrelation time constant as that determined from speckle intensity autocorrelation - a popular conventional measure of scatterer movement. We investigated this decorrelation characteristic time through a 1.5-mm-thick dorsal skin flap of a living mouse and found that it ranges from 50 ms to 2.5 s depending on the level of immobilization. This study provides information on relevant time scales for applying OPC to living tissues.

Upconversion nanoparticles: a versatile solution to multiscale biological imaging.

Lanthanide-doped photon upconverting nanomaterials are emerging as a new class of imaging contrast agents, providing numerous unprecedented possibilities in the realm of biomedical imaging. Because of their ability to convert long-wavelength near-infrared excitation radiation into shorter-wavelength emissions, these nanomaterials are able to produce assets of low imaging background, large anti-Stokes shift, as well as high optical penetration depth of light for deep tissue optical imaging or light-activated drug release and therapy. The aim of this review is to line up some issues associated with conventional fluorescent probes, and to address the recent advances of upconverting nanoparticles (UCNPs) as a solution to multiscale biological imaging applications.

Up-converting NaYF4:0.1%Tm3+, 20%Yb3+ nanoparticles as luminescent labels for deep-tissue optical imaging

Optical imaging plays an important role in biomedical research being extremely useful for early detection, screening and image-guided therapy. Lanthanide-doped up-converting nanoparticles were ideally suited for bioimaging because they could be excited in near infrared (NIR) and emit in NIR or visible (VIS). Here, we compared lanthanide doped up-converting NaYF4 and organic fluorophores for application in deep-tissue imaging. For that purpose - tissue phantoms mimicking the natural properties of light scattering by living tissues were prepared. The studies allowed to quantitatively compare optical resolution of different fluorescent compounds, revealing that the NIR photoexcitation was favorable over conventional UV photoexcitation.

Ultrasonically encoded wavefront shaping for focusing into random media.

Phase distortions due to scattering in random media restrict optical focusing beyond one transport mean free path. However, scattering can be compensated for by applying a correction to the illumination wavefront using spatial light modulators. One method of obtaining the wavefront correction is by iterative determination using an optimization algorithm. In the past, obtaining a feedback signal required either direct optical access to the target region, or invasive embedding of molecular probes within the random media. Here, we propose using ultrasonically encoded light as feedback to guide the optimization dynamically and non-invasively. In our proof-of-principle demonstration, diffuse light was refocused to the ultrasound focal zone, with a focus-to-background ratio of more than one order of magnitude after 600 iterations. With further improvements, especially in optimization speed, the proposed method should find broad applications in deep tissue optical imaging and therapy.

Nanothermometry: All‐In‐One Optical Heater‐Thermometer Nanoplatform Operative From 300 to 2000 K Based on Er3+ Emission and Blackbody Radiation (Adv. Mater. 35/2013)

A nanoplatform accomplishes simultaneous laser-induced heat generation by gold nanoparticles and temperature sensing by (Gd,Yb,Er)2O3 nanorods, presenting considerable potential for hyperthermia and deep-tissue optical imaging. On page 4868, Luis D. Carlos, J. Rocha and co-workers assess the local temperature from the Boltzmann distribution of the Er3+ up-conversion 2H11/2→4|15/2/4S3/2→4|15/2 intensity ratio (300–1050 K) or Planck's law for white-light emission (1200–2000 K).

Investigation of Surface Plasmon Resonance in Multilayered Onion-Like Heteronanocrystal Structures

This paper investigates the interaction of light with some novel multilayered nanostructure in a nonabsorbing medium, which causes change in optical features such as plasmon resonances and their hybridization. The latter phenomenon provides a powerful means of confining light to multilayered nanostructures interfaces, which can generate intense local electromagnetic fields and significantly can amplify the signal. Especially, the motivation of this paper is to determine some suitable nanoparticle geometries and their composition that would result to plasmon tunability (plasmon hybridization) across the visible to near infrared spectra. With plasmons, photonic signals can be manipulated on the nanoscale and can be used in biomedical applications such as high-resolution optical imaging, electromagnetic field enhancement, and biosensing. Moreover, it can be used not only for the enhancement of electromagnetic fields in deep-tissue optical imaging but also in the two-operational distinct spectra for multicolor optical imaging. Specifically, we have investigated some nanoparticles with a different composition that has a strong plasmon resonance which extend into considering spectra (infrared regions). Furthermore, the splitting of the surface plasmon into two distinctive modes as a result of the difference in polarization between the nanoparticle's outer and inner surfaces has been probed. Finally, some simulations are done and their results are shown that the plasmon resonance of the concentric novel nanostructures is due to the hybridization of the inner and outer shell plasmons.

Effects of nanoencapsulation and PEGylation on biodistribution of indocyanine green in healthy mice: quantitative fluorescence imaging and analysis of organs

Near-infrared nanoconstructs present a potentially effective platform for site-specific and deep tissue optical imaging and phototherapy. We have engineered a polymeric nanocapsule composed of polyallylamine hydrochloride (PAH) chains cross-linked with sodium phosphate and doped with indocyanine green (ICG) toward such endeavors. The ICG-doped nanocapsules were coated covalently with polyethylene glycol (5000 daltons) through reductive amination. We administrated the constructs by tail vein injection to healthy mice. To characterize the biodistribution of the constructs, we performed in vivo quantitative fluorescence imaging and subsequently analyzed the various extracted organs. Our results suggest that encapsulation of ICG in these PEGylated constructs is an effective approach to prolong the circulation time of ICG and delay its hepatic accumulation. Increased bioavailability of ICG, due to encapsulation, offers the potential of extending the clinical applications of ICG, which are currently limited due to rapid elimination of ICG from the vasculature. Our results also indicate that PAH and ICG-doped nanocapsules (ICG-NCs) are not cytotoxic at the levels used in this study.

Deep tissue optical imaging of upconverting nanoparticles enabled by exploiting higher intrinsic quantum yield through use of millisecond single pulse excitation with high peak power

We have accomplished deep tissue optical imaging of upconverting nanoparticles at 800 nm, using millisecond single pulse excitation with high peak power. This is achieved by carefully choosing the pulse parameters, derived from time-resolved rate-equation analysis, which result in higher intrinsic quantum yield that is utilized by upconverting nanoparticles for generating this near infrared upconversion emission. The pulsed excitation approach thus promises previously unreachable imaging depths and shorter data acquisition times compared with continuous wave excitation, while simultaneously keeping the possible thermal side-effects of the excitation light moderate. These key results facilitate means to break through the general shallow depth limit of upconverting-nanoparticle-based fluorescence techniques, necessary for a range of biomedical applications, including diffuse optical imaging, photodynamic therapy and remote activation of biomolecules in deep tissues.

Multispectral optoacoustic tomography of myocardial infarction

ObjectivesTo investigate the feasibility of a high resolution optical imaging strategy for myocardial infarction. BackgroundNear-infrared approaches to imaging cardiovascular disease enable visualization of disease-associated biological processes in vivo. However, even at the scale of small animals, the strong scattering of light prevents high resolution imaging after the first 1–2mm of tissue, leading to degraded signal localization. MethodsMultispectral optoacoustic tomography (MSOT) was used to non-invasively image myocardial infarction (MI) in a murine model of coronary artery ligation at resolutions not possible with current deep-tissue optical imaging methods. Post-MI imaging was based on resolving the spectral absorption signature of a dendritic polyglycerol sulfate-based (dPGS) near-infrared imaging agent targeted to P- and L-selectin. ResultsIn vivo imaging succeeded in detection of the agent in the injured myocardium after intravenous injection. The high anatomic resolution (<200μm) achieved by the described method allowed signals originating in the infarcted heart to be distinguished from uptake in adjacent regions. Histological analysis found dPGS signal in infarcted areas, originating from leukocytes and endothelial cells. ConclusionsMSOT imaging of myocardial infarction provides non-invasive visualization of optical contrast with a high spatial resolution that is not degraded by the scattering of light.