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Two-photon microscopy (2PM) has brought unique insight into the mechanisms underlying immune system dynamics and function since it enables monitoring of cellular motility and communication in complex systems within their genuine environment-the living organism. However, use of 2PM in clinical settings is limited. In contrast, optical coherence tomography (OCT), a noninvasive label-free diagnostic imaging method, which allows monitoring morphologic changes of large tissue regions in vivo, has found broad application in the clinic. Here we developed a combined multimodal technology to achieve near-instantaneous coregistered OCT, 2PM, and second harmonic generation (SHG) imaging over large volumes (up to 1,000 × 1,000 × 300 μm3 ) of tendons and other tissue compartments in mouse paws, as well as in mouse lymph nodes, spleens, and femurs. Using our multimodal imaging approach, we found differences in macrophage cell shape and motility behavior depending on whether they are located in tendons or in the surrounding tissue compartments of the mouse paw. The cellular shape of tissue-resident macrophages, indicative for their role in tissue, correlated with the supramolecular organization of collagen as revealed by SHG and OCT. Hence, the here-presented approach of coregistered OCT and 2PM has the potential to link specific cellular phenotypes and functions (as revealed by 2PM) to tissue morphology (as highlighted by OCT) and thus, to build a bridge between basic research knowledge and clinical observations. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

The adult mammary gland undergoes dynamic changes during puberty and the postnatal developmental cycle. The mammary epithelium is composed of a bilayer of outer basal, or myoepithelial, cells and inner luminal cells, the latter lineage giving rise to the milk‐producing alveolar cells during pregnancy. These luminal alveolar cells undergo Stat3‐mediated programmed cell death following the cessation of lactation. It is established that immune cells in the microenvironment of the gland have a role to play both in the ductal outgrowth during puberty and in the removal of dead cells and remodelling of the stroma during the process of postlactational regression. However, most studies have focussed on the role of the stromal immune cell compartment or have quantified immune cell populations in tissue extracts. Our recent development of protocols for deep imaging of the mammary gland in three dimensions (3D) has enabled the architectural relationship between immune cells and the epithelium to be examined in detail, and we have discovered a surprisingly dynamic relationship between the basal epithelium and leucocytes. Furthermore, we have observed morphological changes in the myoepithelial cells, as involution progresses, which were not revealed by previous work in 2D tissue sections and whole tissue. This dynamic architecture suggests a role for myoepithelial cells in the orderly progression of involution. We conclude that deep imaging of mammary gland and other tissues is essential for analysing complex interactions between cellular compartments., In this study by Christine Watson and colleagues, deep 3D‐imaging was used to investigate changes in the architecture of myoepithelial cells and leukocytes in mammary ducts and lobuloalveolar structures. Leukocytes intercalate between ductal myoepithelial and luminal cells and surprisingly, closely associate with myoepithelial cells during lactation and involution, adopting their shape. Myoepithelial cells contract progressively as involution proceeds and shrink back towards the ducts, possibly providing a barrier to the dissemination of milk and dying luminal cells.

Bioluminescent imaging (BLI) technology has been extensively applied due to various advantages such as noninvasiveness, high sensitivity and selectivity, excellent biocompatibility and real-time visualization and monitoring. The firefly luciferase (Fluc)/luciferin system, one of the principal bioluminescent systems, has been developed as a sensor for imaging biological processes. However, a limited number of Fluc substrates hamper the further application of firefly luciferase/luciferin systems for biomedical purposes. Here we describe an approach to synthesize a series of novel luciferin substrates (cyaLucs) that produced elevated bioluminescent signals in vitro. Furthermore, we demonstrate the high efficiency of N-cyclobutylaminoluciferin (cybLuc) with high light emission and long duration in deep tissue imaging by diagnosis of cerebral tumors in vivo in a rodent model.

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.

Photoacoustic tomography has been developed for in vivo functional, metabolic, molecular, and histologic imaging by physically combining optical and ultrasonic waves. Broad applications include early-cancer detection and brain imaging. High-resolution optical imaging—such as confocal microscopy, two-photon microscopy, and optical coherence tomography—is limited to superficial imaging within the optical diffusion limit (~1 mm in the skin) of the surface of scattering tissue. By synergistically combining light and sound, photoacoustic tomography provides deep penetration at high ultrasonic resolution and high optical contrast.

This feature issue of Biomedical Optics Express represents a cross-section of the most recent work in tissue optics, including exciting developments in tissue optical clearing, deep tissue imaging, optical elastography, nanophotonics in tissue, and therapeutic applications of light, amongst others. A collection of 33 papers provides a comprehensive overview of current research in tissue optics, much of it inspired and informed by the pioneering work of Prof. Valery Tuchin. The issue contains three invited manuscripts and several mini-reviews that we hope will benefit researchers in this exciting area.

The making of new nanomaterials for deep tissue imaging is important for clinic applications. In this work, Erbium based metal organic complex nanoparticles is synthesized. They show near-infrared fluorescence (800-1100 nm) when excited by 785 nm light. Deep tissues imaging is demonstrated using a nanoparticle-loaded 2 mL centrifuge tube covered with pig bacon meat tissue. The maximum imaging depth larger than 1.4 cm is achieved. Our work provides a validated procedure to make nanoparticles for near infrared deep tissue imaging.

Summary The gastric corpus epithelium is the thickest part of the gastrointestinal tract and is rapidly turned over. Several markers have been proposed for gastric corpus stem cells in both isthmus and base regions. However, the identity of isthmus stem cells (IsthSCs) and the interaction between distinct stem cell populations is still under debate. Here, based on unbiased genetic labeling and biophysical modeling, we show that corpus glands are compartmentalized into two independent zones, with slow-cycling stem cells maintaining the base and actively cycling stem cells maintaining the pit-isthmus-neck region through a process of “punctuated” neutral drift dynamics. Independent lineage tracing based on Stmn1 and Ki67 expression confirmed that rapidly cycling IsthSCs maintain the pit-isthmus-neck region. Finally, single-cell RNA sequencing (RNA-seq) analysis is used to define the molecular identity and lineage relationship of a single, cycling, IsthSC population. These observations define the identity and functional behavior of IsthSCs., Graphical Abstract, Highlights • Marker-free lineage tracing reveals two types of stem cells in the gastric corpus • Actively cycling isthmus stem cells follow “punctuated” neutral drift dynamics • Stmn1 and Ki67 lineage tracing confirms the active cycling of isthmus stem cells • Single-cell RNA-seq defines identity and lineage relationship of isthmus stem cells, Using lineage-tracing assays and single-cell gene expression profiling, Han et al. show that the mouse gastric corpus gland is compartmentalized, with base and isthmus regions supported by two separate independent stem cell populations. Isthmus stem cells are rapidly cycling, with a broad expression signature, and undergo “punctuated” neutral drift dynamics.

Recently, tremendous progress in synthetic micro/nanomotors in diverse environment has been made for potential biomedical applications. However, existing micro/nanomotor platforms are inefficient for deep tissue imaging and motion control in vivo. Here, we present a photoacoustic computed tomography (PACT) guided investigation of micromotors in intestines in vivo. The micromotors enveloped in microcapsules are stable in the stomach and exhibit efficient propulsion in various biofluids once released. The migration of micromotor capsules toward the targeted regions in intestines has been visualized by PACT in real time in vivo. Near-infrared light irradiation induces disintegration of the capsules to release the cargo-loaded micromotors. The intensive propulsion of the micromotors effectively prolongs the retention in intestines. The integration of the newly developed microrobotic system and PACT enables deep imaging and precise control of the micromotors in vivo and promises practical biomedical applications, such as drug delivery.

Computational imaging in combination with full-field multiphoton microscopy demonstrates rapid in vivo deep tissue imaging., Nonlinear optical microscopy has enabled in vivo deep tissue imaging on the millimeter scale. A key unmet challenge is its limited throughput especially compared to rapid wide-field modalities that are used ubiquitously in thin specimens. Wide-field imaging methods in tissue specimens have found successes in optically cleared tissues and at shallower depths, but the scattering of emission photons in thick turbid samples severely degrades image quality at the camera. To address this challenge, we introduce a novel technique called De-scattering with Excitation Patterning or “DEEP,” which uses patterned nonlinear excitation followed by computational imaging–assisted wide-field detection. Multiphoton temporal focusing allows high-resolution excitation patterns to be projected deep inside specimen at multiple scattering lengths due to the use of long wavelength light. Computational reconstruction allows high-resolution structural features to be reconstructed from tens to hundreds of DEEP images instead of millions of point-scanning measurements.

We present three-dimensional (3-D) high-resolution spectral-domain optical coherence microscopy (SD-OCM) by using a supercontinuum (SC) fiber laser source with 300-nm spectral bandwidth (full-width at half-maximum) in the 1700-nm spectral band. By using low-coherence interferometry with SC light and a confocal detection scheme, we realized lateral and axial resolutions of 3.4 and 3.8 μm in tissue (n=1.38), respectively. This is, to the best of our knowledge, the highest 3-D spatial resolution reported among those of Fourier-domain optical coherence imaging techniques in the 1700-nm spectral band. In our SD-OCM, to enhance the imaging depth, a full-range method was implemented, which suppressed the formation of a coherent ghost image and allowed us to set the zero-delay position inside the samples. We demonstrated the 3-D high-resolution imaging capability of 1700-nm SD-OCM through the measurement of an interference signal from a mirror surface and imaging of a single 200-nm polystyrene bead and a pig thyroid gland. Deep tissue imaging at a depth of up to 1.8 mm was also demonstrated. This is the first demonstration of 3-D high-resolution SD-OCM in the 1700-nm spectral band.

A new donor (D)-acceptor (A) type naphthalene-based oxazepine-containing fluorophore, OXN-1, is reported, which shows unusually high stability in various environments. Its photophysical properties and structural stabilities under harsh conditions are thoroughly examined. The high stability of OXN-1 is explained by quantum chemical calculations. Its exceptional bioimaging capabilities for cells with low cytotoxicity are verified. In addition, its deep tissue imaging ability with two-photon microscopy (TPM) is evaluated.

A boron-dipyrromethene (BODIPY) dye emitting in the near-infrared (NIR) I region (723 nm) exhibits strong saturable absorption at 680 nm. Its multiphoton absorption spectra in the NIR II and III regions are determined. Three-photon fluorescence imaging of the BODIPY-labeled cells excited at 1665 nm is also demonstrated.

Photoacoustic (PA) imaging in the second near-infrared (NIR-II) window (especially at 1064 nm) has the benefits of low background signal, high spatial resolution and deep tissue penetration. Here we report a semiconducting polymer nanoparticle (PDPPTBZ NP) and demonstrate its potential as a contrast agent for PA imaging of orthotopic brain tumors, using a 1064 nm pulsed laser as a light source. PDPPTBZ NPs have maximum absorption at 1064 nm with a mass extinction coefficient of 43 mL mg−1 cm−1, which is the highest value reported so far in this region. The high photothermal conversion efficiency (67%) and near non-fluorescence impart PDPPTBZ NPs with excellent PA properties. We used PDPPTBZ NP-containing agar gel phantoms even at a low concentration (50 μg mL−1) to successfully image to a depth of 4 cm (of chicken-breast tissue), with an ultralow power fluence (4 mJ cm−2). Furthermore, we could clearly visualize a glioma tumor in a mouse at a depth of 3.8 mm below the skull. This study demonstrates that PDPPTBZ NPs display great potential as a NIR-II PA contrast agent for high quality deep tissue imaging.

Wireless nano-/micromotors powered by chemical reactions and/or external fields generate motive forces, perform tasks, and significantly extend short-range dynamic responses of passive biomedical microcarriers. However, before micromotors can be translated into clinical use, several major problems, including the biocompatibility of materials, the toxicity of chemical fuels, and deep tissue imaging methods, must be solved. Nanomaterials with enzyme-like characteristics (e.g., catalase, oxidase, peroxidase, superoxide dismutase), that is, nanozymes, can significantly expand the scope of micromotors' chemical fuels. A convergence of nanozymes, micromotors, and microfluidics can lead to a paradigm shift in the fabrication of multifunctional micromotors in reasonable quantities, encapsulation of desired subsystems, and engineering of FDA-approved core-shell structures with tuneable biological, physical, chemical, and mechanical properties. Microfluidic methods are used to prepare stable bubbles/microbubbles and capsules integrating ultrasound, optoacoustic, fluorescent, and magnetic resonance imaging modalities. The aim here is to discuss an interdisciplinary approach of three independent emerging topics: micromotors, nanozymes, and microfluidics to creatively: 1) embrace new ideas, 2) think across boundaries, and 3) solve problems whose solutions are beyond the scope of a single discipline toward the development of micro-bio-chemo-mechanical-systems for diverse bioapplications.

Visualization of biological targets such as crucial cells and biomolecules in living subjects is critical for the studies of important biological processes. Though 1 H magnetic resonance imaging (MRI) has demonstrated its power in offering detailed anatomical and pathological information, its capacity for in vivo tracking of biological targets is limited by the high biological background of 1 H. 19 F distinguishes itself from its competitors as an exceptional complement to 1 H in MRI through its high sensitivity, low biological background, and broad chemical shift range. The specificity and sensitivity of 19 F MRI can be further boosted with activatable nanoprobes. The advantages of 19 F MRI with activatable nanoprobes enable in vivo detection and imaging at the cellular or even molecular level in deep tissues, rendering this technique appealing as a potential solution for visualization of biological targets in living subjects. Here, recent progress over the past decades on activatable 19 F MRI nanoprobes made from three major 19 F-containing compounds, as well as present challenges and potential opportunities, are summarized to provide a panoramic prospective for the people who are interested in this emerging and exciting field.

Glycerol–water mediated convenient synthesis of PbS quantum dots (QDs) is introduced utilizing distinctive precipitation strategies. It is exceptionality fascinating to observe that the aforementioned factor assumes a major role in the capping process, stability as well as purity and crystalline nature of the QD. Strong red fluorescence from QDs in the HeLa cell makes these materials suitable for deep tissue imaging.

As the control center of organisms, the brain remains little understood due to its complexity. Taking advantage of imaging methods, scientists have found an accessible approach to unraveling the mystery of neuroscience. Among these methods, optical imaging techniques are widely used due to their high molecular specificity and single-molecule sensitivity. Here, we overview several optical imaging techniques in neuroscience of recent years, including brain clearing, the micro-optical sectioning tomography system, and deep tissue imaging.

Over the past decade, there have been extensive developments in the field of lanthanide-based nanoparticles (NPs). Most studies have focused on the application of upconverting NaYF4-based NPs for deep tissue imaging and paramagnetic NaGdF4 NPs for MRI. Current applications for the remaining members of the lanthanide series are rather limited. Recently, a novel bioanalytical technique known as mass cytometry (MC) has been developed which can benefit from the entire lanthanide series of NPs. MC is a high-throughput multiparametric cell-by-cell analysis technique based on atomic mass spectrometry that uses antibodies labeled with metal isotopes for biomarker detection. NaLnF4 NPs offer the promise of high sensitivity coupled with multiparameter detection, provided that NPs can be synthesized with a narrow size distribution. Here we describe the synthesis of six members of this NP family (NaSmF4, NaEuF4, NaGdF4, NaTbF4, NaDyF4, NaHoF4) with the appropriate size (5–30 nm) and size distribution (CV < 5%) for MC...

Near-infrared (NIR) chemiluminescence (CL) emission is highly favorable for deep-tissue imaging, but chemically conjugated NIR CL emitters with the aggregation-induced emission (AIE) property for biotechnology are seldom reported. Herein, an AIE-active NIR CL emitter, TBL, is synthesized by conjugating luminol unit with electron-accepting benzothiadiazole and an electron-donating triphenylamine, and subsequently TBL dots are prepared by using F127 as the surfactant. The CL emission of TBL dots can last continuously for over 60 min and can be employed for quantitative (in vitro) and qualitative (in vivo) detection of 1 O2 . Strikingly, the NIR CL emission can penetrate through tissues with a total thickness of over 3 cm, exhibiting significantly better performance than NIR fluorescence emission and blue CL emission. Moreover, the successful differentiation of tumor and normal tissues by TBL-based CL imaging in vivo also paves the way for CL-guided cancer diagnosis and surgery.

There has been widespread interest in the application of simple light-based methods and optical imaging as adjunctive tools in oral oncology. These optical imaging techniques exploit differences in properties such as absorption, reflectance, and light scattering between normal and transformed epithelium. Optical imaging methods can also utilize tissue autofluorescence arising from endogenous chromatophores to detect malignant tissue. For example, early oral malignancy is often associated with a loss of fluorescence or fluorescence visualization loss (FVL) which may be used to aid in tissue selection for biopsy. The autofluorescence-based Visual Enhanced Light scope (VELscope®), chemiluminescence-based ViziLite® system, the Identafi® system that uses multispectral fluorescence and reflectance, and narrow band imaging (NBI) instruments are among the optical imaging-based diagnostic platforms that are currently available for clinical use. In addition, photoacoustic imaging (PAI) is an advanced hybrid imaging method that allows for deep tissue imaging and is actively being evaluated for diagnostic applications in oncology. In this chapter, we will review the basics of these optical imaging methods and summarize preclinical and clinical evidence on their performance in oral oncology. The goal of this chapter is to provide the reader with an overview of these methods and their potential clinical applications.

Imaging of small animals has played an indispensable role in preclinical research by providing high dimensional physiological, pathological, and phenotypic insights with clinical relevance. Yet pure optical imaging suffers from either shallow penetration (up to ~1–2 mm) or a poor depth-to-resolution ratio (~3), and non-optical techniques for whole-body imaging of small animals lack either spatiotemporal resolution or functional contrast. A stand-alone single-impulse photoacoustic computed tomography (PACT) system has been built, which successfully mitigates these limitations by integrating high spatiotemporal resolution, deep penetration, and full-view fidelity, as well as anatomical, dynamical, and functional contrasts. Based on hemoglobin absorption contrast, the whole-body dynamics and large scale brain functions of rodents have been imaged in real time. The absorption contrast between cytochrome and lipid has enabled PACT to resolve MRI-like whole brain structures. Taking advantage of the distinct absorption signature of melanin, unlabeled circulating melanoma cells have been tracked in real time in vivo. Assisted by near-infrared dyes, the perfusion processes have been visualized in rodents. By localizing single-dyed droplets, the spatial resolution of PACT has been improved by six-fold in vivo. The migration of metallic-based microrobots toward the targeted regions in the intestines has been monitored in real time. Genetically encoded photochromic proteins benefit PACT in detection sensitivity and specificity. The unique photoswitching characteristics of different photochromic proteins allow quantitative multi-contrast imaging at depths. A split version of the photochromic protein has permitted PA detection of protein-protein interactions in deep-seated tumors. The photochromic behaviors have also been utilized to guide photons to form an optical focus inside live tissue. As a rapidly evolving imaging technique, PACT promises pre-clinical applications and clinical translation.

Adaptive optics (AO) has been widely used in optical microscopy to recover high-resolution images in deep tissue. However, in conventional AO systems, the corrected field of view (FOV) of a single guide star is usually quite limited. Here we demonstrate a conjugate AO system based on automatic optimal multiple guide stars selection algorithm to achieve large effective corrected FOV with a small number of guide stars. For a random phase mask as the scattering medium, the effective correction coverage ratio can be increased to ∼ 5.09 times than that in a conventional CAO system. For a mouse brain slice with 117 μ m thickness, the effective corrected FOV is larger than that of conventional CAO system by a factor of ∼ 2.58. Therefore, our method shows potentials in aberration correction with large FOV for deep tissue imaging

Photoacoustic imaging is a noninvasive imaging technique having the advantages of high-optical contrast and good acoustic resolution at improved imaging depths. Light transport in biological tissues is mainly characterized by strong optical scattering and absorption. Photoacoustic microscopy is capable of achieving high-resolution images at greater depth compared to conventional optical microscopy methods. In this work, we have developed a high-resolution, acoustic resolution photoacoustic microscopy (AR-PAM) system in the near infra-red (NIR) window II (NIR-II, eg, 1064 nm) for deep tissue imaging. Higher imaging depth is achieved as the tissue scattering at 1064 nm is lesser compared to visible or near infrared window-I (NIR-I). Our developed system can provide a lateral resolution of 130 mu m, axial resolution of 57 mu m, and image up to 11 mm deep in biological tissues. This 1064-AR-PAM system was used for imaging sentinel lymph node and the lymph vessel in rat. Urinary bladder of rat filled with black ink was also imaged to validate the feasibility of the developed system to study deeply seated organs. The authors would like to acknowledge the financial support from Tier 2 grant funded by the Singapore Ministry of Education Tier‐2 Academic Research Funds (ARC2/15: M4020238). The authors would like to acknowledge Dr Rhonnie Austria Dienzo for his help with animal experiments. peer-reviewed 2019-12-03

In this work, a near-infrared probe-based confocal microendoscope (pCM) with a 785 nm laser source, a long working distance, and a probe with diameter of 2.6 mm that can be compatible with a conventional endoscope is demonstrated to produce deep-tissue images at cellular resolutions with enhanced contrast and signal-to-noise ratio. Theoretical simulations and experiments confirm that near-infrared light can optimize the image quality. Abundant details of mouse esophagus obtained at different depths demonstrate the system’s ability to image deep tissues at cellular resolutions, which makes it possible to diagnose diseases in the digestive tract in real time, laying a solid foundation for clinical applications.

Bioimaging probes, which can emit near-infrared (NIR) light and are excitable with NIR light, are promising for deep tissue imaging in vivo. Lanthanide-doped phosphors, such as Y2O3:Tm,Yb; Y2O3:Ho,Yb; and Y2O3:Er,Yb, are candidates for the probes. We enhanced the NIR emission of the three kinds of lanthanide-doped phosphor by using Li-ion doping. The probes were applied to cellular imaging.

In biomedical imaging, all optical techniques face a fundamental trade-off between spatial resolution and tissue penetration. Therefore, obtaining an organelle-level resolution image of a whole organ has remained a challenging and yet appealing scientific pursuit. Over the past decade, optical microscopy assisted by mechanical sectioning or chemical clearing of tissue has been demonstrated as a powerful technique to overcome this dilemma, one of particular use in imaging the neural network. However, this type of techniques needs lengthy special preparation of the tissue specimen, which hinders broad application in life sciences. Here, we propose a new label-free three-dimensional imaging technique, named microtomy-assisted photoacoustic microscopy (mPAM), for potentially imaging all biomolecules with 100% endogenous natural staining in whole organs with high fidelity. We demonstrate the first label-free mPAM, using UV light for label-free histology-like imaging, in whole organs (e.g., mouse brains), most of them formalin-fixed and paraffin- or agarose-embedded for minimal morphological deformation. Furthermore, mPAM with dual wavelength illuminations is also employed to image a mouse brain slice, demonstrating the potential for imaging of multiple biomolecules without staining. With visible light illumination, mPAM also shows its deep tissue imaging capability, which enables less slicing and hence reduces sectioning artifacts. mPAM could potentially provide a new insight for understanding complex biological organs.

Photoacoustic (PA) imaging is a biomedical imaging method that can provide both structural and functional information of living tissues beyond the optical diffusion limit by combining the concepts of conventional optical and ultrasound imaging methods. Although endogenous chromophores can be utilized to acquire PA images of biological tissues, exogenous contrast agents that absorb near-infrared (NIR) lights have been extensively explored to improve the contrast and penetration depth of PA images. Here, we demonstrate Bi2Se3 nanoplates, that strongly absorbs NIR lights, as a contrast agent for PA imaging. In particularly, the Bi2Se3 nanoplates produce relatively strong PA signals with an optical wavelength of 1064 nm, which has several advantages for deep tissue imaging including: (1) relatively low absorption by other intrinsic chromophores, (2) cost-effective light source using Nd:YAG laser, and (3) higher available energy than other NIR lights according to American National Standards Institute (ANSI) safety limit. We have investigated deep tissue imaging capability of the Bi2Se3 nanoplates by acquiring in vitro PA images of microtubes under chicken breast tissues. We have also acquired in vivo PA images of bladders, gastrointestinal tracts, and sentinel lymph nodes in mice after injection of the Bi2Se3 nanoplates to verify their applicability to a variety of biomedical research. The results show the promising potential of the Bi2Se3 nanoplates as a PA contrast agent for deep tissue imaging with an optical wavelength of 1064 nm.

There is increasing interest in the three-dimensional visualization and quantification of cellular circuits in the brain and therefore optical clearing methods are highly in demand for brain imaging. In particular, clarification without membrane damage is required to image lipophilic tracer-labeled neural tracts. However, previously reported DiI-compatible optical clearing methods are relatively slow and can hinder transparency for imaging. Here, we present DAS, a new, convenient, inexpensive and reproducible aqueous clearing reagent that can efficiently clarify tissues with minimal volume enlargement and reliably preserves emission from fluorescent proteins and lipophilic dyes in membrane integrity preserved tissues.

We demonstrate a multimodal system integrating optical coherence tomography (OCT) with wavelength modulated spatially offset Raman spectroscopy. This has the capability to detect co-registered Raman and OCT signals from targets deeply embedded in tissue.

Existing brain imaging sensors are either limited in their depth, resolution, and/or inflict trauma. To improve on these limitations, we demonstrate fluorescent microscopy through an optical cannula for deep tissue imaging.

We demonstrate a new approach, temporal focusing microscopy with single-pixel detection (TEMPPIX), for wide-field multiphoton imaging through scattering media without any a priori knowledge or requirement to determine the properties of the media.

Biological tissue is a highly scattering medium that prevents deep imaging of light. For medical applications, optical imaging offers a molecular sensitivity that would be beneficial for diagnosing and monitoring of diseases. Acousto-optical tomography has the molecular sensitivity of optical imaging with the resolution of ultrasound and has the potential for deep tissue imaging. Here, we present a theoretical study of a system that combines acousto-optical tomography and slow light spectral filters created using spectral hole burning methods. Using Monte Carlo simulations, a model to obtain the contrast-to-noise ratio (CNR) deep in biological tissue was developed. The simulations show a CNR > 1 for imaging depths of ∼5 cm in a reflection mode setup, as well as, imaging through ∼12 cm in transmission mode setups. These results are promising and form the basis for future experimental studies.

Multiple scattering still remains as the ultimate barrier for in-vivo deep tissue imaging. Here, we will discuss our efforts to overcome multiple scattering and enable diffraction limited, or even subwavelength resolution imaging through highly scattering media including living biological tissue.

Innate and adaptive immune interactions within the central nervous system (CNS) and surrounding meninges contribute significantly to neural homeostasis as well as a variety of different neurological disorders. Two-photon laser scanning microscopy is a deep tissue imaging technique that provides a means to image immune cell dynamics and interactions in the living CNS with high spatial and temporal resolution. Optical access to the brain and meninges can be achieved through the creation of thinned skull windows, which can be made without inducing damage and inflammation in the underlying tissue. This protocol provides guidance on how to create a thinned skull window without causing CNS injury. We also describe a highly reproducible method to induce a mild traumatic brain injury using the thinned skull approach. © 2018 by John Wiley & Sons, Inc.

Fluorescent carbonaceous dots (CDs) have attracted much attention due to their unique properties. However, their application in non-invasive imaging of diseased tissues was restricted by the short excitation/emission wavelength and the poor targeting efficiency of CDs. In this study, CDs were prepared from sucrose and glutamic acid with a particle size of 57.5 nm. An obvious emission could be observed at 600 nm to 700 nm when excited at around 500 nm. This property enabled CDs with a capacity for deep tissue imaging with low background adsorption. RGD, a ligand which could target most tumor and neovasculature cells, was anchored onto CDs after PEGylation. The product, RGD–PEG–CDs could accumulate in MCF-7/ADR xenografts at high intensity, which was 1.65-fold higher than that of PEG–CDs. Furthermore, RGD–PEG–CDs showed good colocalization with neovasculature. Thus, RGD–PEG–CDs could be used for non-invasive MCF-7/ADR tumor imaging. CDs functionalized with other ligands may also be used as a non-invasive probe for many kinds of tumor imaging.

As one of the most powerful tools to visualize PPIs in living cells, bimolecular fluorescence complementation (BiFC) has gained great advancement during recent years, including deep tissue imaging with far-red or near-infrared fluorescent proteins or super-resolution imaging with photochromic fluorescent proteins. However, little progress has been made toward simultaneous detection and visualization of multiple PPIs in the same cell, mainly due to the spectral crosstalk. In this report, we developed novel BiFC assays based on large-Stokes-shift fluorescent proteins (LSS-FPs) to detect and visualize multiple PPIs in living cells. With the large excitation/emission spectral separation, LSS-FPs can be imaged together with normal Stokes shift fluorescent proteins to realize multicolor BiFC imaging using a simple illumination scheme. We also further demonstrated BiFC rainbow combining newly developed BiFC assays with previously established mCerulean/mVenus-based BiFC assays to achieve detection and visualization of four PPI pairs in the same cell. Additionally, we prove that with the complete spectral separation of mT-Sapphire and CyOFP1, LSS-FP-based BiFC assays can be readily combined with intensity-based FRET measurement to detect ternary protein complex formation with minimal spectral crosstalk. Thus, our newly developed LSS-FP-based BiFC assays not only expand the fluorescent protein toolbox available for BiFC but also facilitate the detection and visualization of multiple protein complex interactions in living cells.

We have developed a new class of two-photon absorbing dyes that are far-red emitting, water-soluble, and very bright inside cells as well as in tissue. The significant autofluorescence from yellow wavelength region in tissue imaging can be addressed by deep-red emitting dyes., The fluorescence imaging of tissue is essential for studying biological events beyond the cellular level. Two-photon microscopy based on the nonlinear light absorption of fluorescent dyes is a viable tool for the high resolution imaging of tissue. A key limitation for deep tissue imaging is the autofluorescence from intrinsic biomolecules. Here, we report a systematic study that discloses relative autofluorescence interference, which is dependent on the type of tissue and the excitation and emission wavelengths in two-photon imaging. Among the brain, kidney, liver, lung, and spleen mouse tissues examined, the kidney tissue exhibited prominent autofluorescence followed by the liver and others. Notably, regardless of the tissue type, prominent autofluorescence is observed not only from the green emission channel but also from the yellow emission channel where common two-photon absorbing dyes also emit, whereas there is minimal autofluorescence from the red channel. The autofluorescence is slightly influenced by the excitation wavelength. Toward minimal autofluorescence, we developed a new class of two-photon absorbing dyes that are far-red emitting, water-soluble, and very bright inside cells as well as in tissue. A comparative assessment of the imaging depth, which is dependent on the three selected dyes that emit in the blue-green, yellow, and far-red regions, shows the importance of far-red emitting dyes for deep tissue imaging.

This paper will present ultra-compact low-cost laser diode sources, including its diode driver electronics, that have been developed for integration in a photoacoustic imaging system. These laser sources deliver mJ pulses in the near-infrared range.

Progress in developing optical imaging for biomedical applications requires customizable and often complex objects known as "phantoms" for testing, evaluation, and calibration. This work demonstrates that 3D printing is an ideal method for fabricating such objects, allowing intricate inhomogeneities to be placed at exact locations in complex or anatomically realistic geometries, a process that is difficult or impossible using molds. We show printed mouse phantoms we have fabricated for developing deep tissue fluorescence imaging methods, and measurements of both their optical and mechanical properties. Additionally, we present a printed phantom of the human mouth that we use to develop an artery localization method to assist in oral surgery.