Noninvasive,high‐speed,near‐infrared imaging of the biomolecular distribution and molecular mechanism of embryonic development in fertilized fish eggs

In this study, the distribution of biomaterials and its molecular mechanism of embryonic development in Japanese medaka fish were analyzed nondestructively and noninvasively without staining using near‐infrared (NIR) imaging. The microscopic NIR imaging system used in this research was a device capable of ultra‐high‐speed imaging; using this system, one can acquire microscopic imaging data in a few seconds. Therefore, the medaka eggs remained alive throughout measurements and were successfully monitored in vivo. The distributions of biomolecules were examined by mapping the intensities of NIR bands resulting from lipids, proteins and water in 2 dimensions (2D). The structures of eyes, lipid bilayer membranes, micelles and water‐structure differences at the interface of different substances constituting different structures on the egg were visualized. Furthermore, insights on the metabolic mechanisms of lipids and membrane functions were drawn from the biased distribution of lipoproteins and the presence of unsaturated fatty acids in the egg membrane. These results indicated the potential for NIR imaging in evaluating the biological functions and metabolic systems of cells and embryos.      

Three‐dimensional cellular imaging in thick biological tissue with confocal detection of one‐photon fluorescence in the near‐infrared II window

Fluorescence imaging in the second near‐infrared optical window (NIR‐II, 900‐1700 nm) has become a technique of choice for noninvasive in vivo imaging in recent years. Greater penetration depths with high spatial resolution and low background can be achieved with this NIR‐II window, owing to low autofluorescence within this optical range and reduced scattering of long wavelength photons. Here, we present a novel design of confocal laser scanning microscope tailored for imaging in the NIR‐II window. We showcase the outstanding penetration depth of our confocal setup with a series of imaging experiments. HeLa cells labeled with PbS quantum dots with a peak emission wavelength of 1276 nm can be visualized through a 3.5‐mm‐thick layer of scattering medium, which is a 0.8% Lipofundin solution. A commercially available organic dye IR‐1061 (emission peak at 1132 nm), in its native form, is used for the first time, as a NIR‐II fluorescence label in cellular imaging. Our confocal setup is capable of capturing optically sectioned images of IR‐1061 labeled chondrocytes in fixed animal cartilage at a depth up to 800 μm, with a superb spatial resolution of around 2 μm.      

Perfusion‐based fluorescence imaging method delineates diverse organs and identifies multifocal tumors using generic near‐infrared molecular probes

Rapid detection of multifocal cancer without the use of complex imaging schemes will improve treatment outcomes. In this study, dynamic fluorescence imaging was used to harness differences in the perfusion kinetics of near‐infrared (NIR) fluorescent dyes to visualize structural characteristics of different tissues. Using the hydrophobic nontumor‐selective NIR dye cypate, and the hydrophilic dye LS288, a high tumor‐to‐background contrast was achieved, allowing the delineation of diverse tissue types while maintaining short imaging times. By clustering tissue types with similar perfusion properties, the dynamic fluorescence imaging method identified secondary tumor locations when only the primary tumor position was known, with a respective sensitivity and specificity of 0.97 and 0.75 for cypate, and 0.85 and 0.81 for LS288. Histological analysis suggests that the vasculature in the connective tissue that directly surrounds the tumor was a major factor for tumor identification through perfusion imaging. Although the hydrophobic dye showed higher specificity than the hydrophilic probe, use of other dyes with different physical and biological properties could further improve the accuracy of the dynamic imaging platform to identify multifocal tumors for potential use in real‐time intraoperative procedures.      

Multispectral photoacoustic imaging of cancer with broadband CuS nanoparticles covering both near‐infrared I and II biological windows

In this study, CuS nanoparticles with optical absorption covering both near‐infrared I (NIR‐I) and NIR‐II biological windows were prepared and served as the contrast agents for multispectral photoacoustic imaging. The physiological parameters including concentrations of deoxyhemoglobin and oxyhemoglobin as well as the water content in the tumor location were quantified based on the multispectral photoacoustic reconstruction method. More importantly, the concentration of CuS nanoparticles/drugs accumulated in the tumor was also recovered after intravenously injection, which are essential for image‐guided cancer theranostics. In addition, phantom and in vivo experimental tests were performed to inspect and compare the imaging depth and signal‐to‐noise ratio (SNR) between the two NIR biological windows. Interestingly, we discovered that a higher SNR was obtained in the NIR‐II window than that in the NIR‐I window. Meanwhile, the multispectral imaging results also demonstrated that the imaging contrast and penetration depth in the NIR‐II window were also significantly improved as compared to those from the NIR‐I window.      

Contact‐free endoscopic photoacoustic sensing using speckle analysis

Photoacoustic endoscopy (PAE) is an emerging imaging modality, which offers a high imaging penetration and a high optical contrast in soft tissue. Most of the developed endoscopic photoacoustic sensing systems use miniaturized contact ultrasound transducers or complex optical approaches. In this work, a new fiber‐based detection technique using speckle analysis for contact‐free signal detection is presented. Phantom and ex vivo experiments are performed in transmission and reflection mode for proof of concept. In summary, the potential of the technique for endoscopic photoacoustic signal detection is demonstrated. The new technique might help in future to broaden the applications of PAE in imaging or guiding minimally invasive laser procedures.      

DNA‐based probes for flow cytometry analysis of endocytosis and recycling

The internalization of proteins plays a key role in cell development, cell signaling and immunity. We have previously developed a specific hybridization internalization probe (SHIP) to quantitate the internalization of proteins and particles into cells. Herein, we extend the utility of SHIP to examine both the endocytosis and recycling of surface receptors using flow cytometry. SHIP was used to monitor endocytosis of membrane‐bound transferrin receptor (TFR) and its soluble ligand transferrin (TF). SHIP enabled measurements of the proportion of surface molecules internalized, the internalization kinetics and the proportion and rate of internalized molecules that recycle to the cell surface with time. Using this method, we have demonstrated the internalization and recycling of holo‐TF and an antibody against the TFR behave differently. This assay therefore highlights the implications of receptor internalization and recycling, where the internalization of the receptor‐antibody complex behaves differently to the receptor‐ligand complex. In addition, we observe distinct internalization patterns for these molecules expressed by different subpopulations of primary cells. SHIP provides a convenient and high throughput technique for analysis of trafficking parameters for both cell surface receptors and their ligands.      

Intraoperative multispectral and hyperspectral label‐free imaging: A systematic review of in vivo clinical studies

Multispectral and hyperspectral imaging (HSI) are emerging optical imaging techniques with the potential to transform the way surgery is performed but it is not clear whether current systems are capable of delivering real‐time tissue characterization and surgical guidance. We conducted a systematic review of surgical in vivo label‐free multispectral and HSI systems that have been assessed intraoperatively in adult patients, published over a 10‐year period to May 2018. We analysed 14 studies including 8 different HSI systems. Current in‐vivo HSI systems generate an intraoperative tissue oxygenation map or enable tumour detection. Intraoperative tissue oxygenation measurements may help to predict those patients at risk of postoperative complications and in‐vivo intraoperative tissue characterization may be performed with high specificity and sensitivity. All systems utilized a line‐scanning or wavelength‐scanning method but the spectral range and number of spectral bands employed varied significantly between studies and according to the system's clinical aim. The time to acquire a hyperspectral cube dataset ranged between 5 and 30 seconds. No safety concerns were reported in any studies. A small number of studies have demonstrated the capabilities of intraoperative in‐vivo label‐free HSI but further work is needed to fully integrate it into the current surgical workflow.      

Molecular mechanisms of non‐transferrin‐bound and transferring‐bound iron uptake in primary hippocampal neurons

The molecular mechanisms of iron trafficking in neurons have not been elucidated. In this study, we characterized the expression and localization of ferrous iron transporters Zip8, Zip14 and divalent metal transporter 1 (DMT1), and ferrireductases Steap2 and stromal cell‐derived receptor 2 in primary rat hippocampal neurons. Steap2 and Zip8 partially co‐localize, indicating these two proteins may function in Fe³⁺ reduction prior to Fe²⁺ permeation. Zip8, DMT1, and Steap2 co‐localize with the transferrin receptor/transferrin complex, suggesting they may be involved in transferrin receptor/transferrin‐mediated iron assimilation. In brain interstitial fluid, transferring‐bound iron (TBI) and non‐transferrin‐bound iron (NTBI) exist as potential iron sources. Primary hippocampal neurons exhibit significant iron uptake from TBI (Transferrin‐⁵⁹Fe³⁺) and NTBI, whether presented as ⁵⁹Fe²⁺‐citrate or ⁵⁹Fe³⁺‐citrate; reductase‐independent ⁵⁹Fe²⁺ uptake was the most efficient uptake pathway of the three. Kinetic analysis of Zn²⁺ inhibition of Fe²⁺ uptake indicated that DMT1 plays only a minor role in the uptake of NTBI. In contrast, localization and knockdown data indicate that Zip8 makes a major contribution. Data suggest also that cell accumulation of ⁵⁹Fe from TBI relies at least in part on an endocytosis‐independent pathway. These data suggest that Zip8 and Steap2 play a major role in iron accumulation from NTBI and TBI by hippocampal neurons.

     

Slide‐free imaging of hematoxylin‐eosin stained whole‐mount tissues using combined third‐harmonic generation and three‐photon fluorescence microscopy

Intraoperative margin assessment of surgical tissues during cancer surgery is clinically important, especially in the case of tissue conserving surgery like Mohs micrographic surgery in which minimization of the surgical area is considered crucial. Frozen pathology is the gold standard of assessing excised tissues for signs of remaining cancerous lesions. The current protocol, however, is time‐consuming and labor‐intensive. Instead of the complex frozen sectioning, staining, and traditional white light microscopy imaging protocol, optically sectioned histopathological imaging of hematoxylin‐eosin stained whole‐mount skin tissues with a subfemtoliter resolution is demonstrated by using nonlinear microscopy in this study. With our proposed method, the reagents of staining and the contrast of imaging are fully consistent with the current clinical standard of frozen pathology, thus facilitating rapid intraoperative assessment of surgical tissues for future applications. Image: Slide‐free nonlinear microscopy imaging of H&E stained whole‐mount skin tissue showing the morphology of sweat glands.      

Two‐photon excitation and direct emission from S2 state of U.S. Food and Drug Administration approved near‐infrared dye: Application of anti‐Kasha's rule for two‐photon fluorescence imaging

In recent years, two‐photon fluorescence microscopy has gained significant interest in bioimaging. It allows the visualization of deeply buried inhomogeneities in tissues. The near‐infrared (NIR) dyes are also used for deep tissue imaging. Indocyanine green (ICG) is the only U.S. Food and Drug Administration (FDA) approved exogenous contrast agent in the NIR region for clinical applications. However, despite its potential candidature, it had never been used as a two‐photon contrast agent for biomedical imaging applications. This letter provides an insight into the scope and application of the two‐photon excitation property of ICG to the second excited singlet (S₂) state in aqueous solution. Furthermore, in this work, we demonstrate the two‐photon cellular imaging application of ICG using direct fluorescence emission from S₂ state for the first time. Our results show that two‐photon excitation to S₂ state of ICG could be achieved with approximately 790 nm wavelength of femtosecond laser, which lies in well‐known “tissue‐optical window.” This property would enable light to penetrate much deeper in the turbid medium such as biological tissues. Thus, ICG could be used as the first FDA approved NIR exogenous contrast agent for two‐photon imaging. These findings can make remarkable influence on preclinical and clinical cell imaging.      

G5G2.5 core‐shell tecto‐dendrimer specifically targets reactive glia in brain ischemia

Secondary neuronal death is a serious stroke complication. This process is facilitated by the conversion of glial cells to the reactive pro‐inflammatory phenotype that induces neurodegeneration. Therefore, regulation of glial activation is a compelling strategy to reduce brain damage after stroke. However, drugs have difficulties to access the CNS , and to specifically target glial cells. In the present work, we explored the use core‐shell polyamidoamine tecto‐dendrimer (G5G2.5 PAMAM ) and studied its ability to target distinct populations of stroke‐activated glial cells. We found that G5G2.5 tecto‐dendrimer is actively engulfed by primary glial cells in a time‐ and dose‐dependent manner showing high cellular selectivity and lysosomal localization. In addition, oxygen‐glucose deprivation or lipopolysaccharides exposure in vitro and brain ischemia in vivo increase glial G5G2.5 uptake; not being incorporated by neurons or other cell types. We conclude that G5G2.5 tecto‐dendrimer is a highly suitable carrier for targeted drug delivery to reactive glial cells in vitro and in vivo after brain ischemia.

     

Single molecule localization deep within thick cells; a novel super‐resolution microscope

A novel 3D imaging system based on single‐molecule localization microscopy is presented to allow high‐accuracy drift‐free (<0.7 nm lateral; 2.5 nm axial) imaging many microns deep into a cell. When imaging deep within the cell, distortions of the point‐spread function result in an inaccurate and very compressed Z distribution. For the system to accurately represent the position of each blink, a series of depth‐dependent calibrations are required. The system and its allied methodology are applied to image the ryanodine receptor in the cardiac myocyte. Using the depth‐dependent calibration, the receptors deep within the cell are spread over a Z range that is many hundreds of nanometers greater than implied by conventional analysis. We implemented a time domain filter to detect overlapping blinks that were not filtered by a stringent goodness of fit criterion. This filter enabled us to resolve the structure of the individual (30 nm square) receptors giving a result similar to that obtained with electron tomography.

High‐accuracy deep imaging of the ryanodine receptor in the cardiac myocyte, using single‐molecule localization microscopy. Depth‐dependent calibrations are performed for accurate depth localization. The optical design featuring two independent and variable focal planes allows real‐time feedback for drift‐free deep imaging.     

Wide‐field color imaging of scatter‐based tissue contrast using both high spatial frequency illumination and cross‐polarization gating

This study characterizes the scatter‐specific tissue contrast that can be obtained by high spatial frequency (HSF) domain imaging and cross‐polarization (CP) imaging, using a standard color imaging system, and how combining them may be beneficial. Both HSF and CP approaches are known to modulate the sensitivity of epi‐illumination reflectance images between diffuse multiply scattered and superficially backscattered photons, providing enhanced contrast from microstructure and composition than what is achieved by standard wide‐field imaging. Measurements in tissue‐simulating optical phantoms show that CP imaging returns localized assessments of both scattering and absorption effects, while HSF has uniquely specific sensitivity to scatter‐only contrast, with a strong suppression of visible contrast from blood. The combination of CP and HSF imaging provided an expanded sensitivity to scatter compared with CP imaging, while rejecting specular reflections detected by HSF imaging. ex vivo imaging of an atlas of dissected rodent organs/tissues demonstrated the scatter‐based contrast achieved with HSF, CP and HSF‐CP imaging, with the white light spectral signal returned by each approach translated to a color image for intuitive encoding of scatter‐based contrast within images of tissue. The results suggest that visible CP‐HSF imaging could have the potential to aid diagnostic imaging of lesions in skin or mucosal tissues and organs, where just CP is currently the standard practice imaging modality.      

Computed tomography for in vivo deep over‐1000 nm near‐infrared fluorescence imaging

This study aims to develop a novel cross‐sectional imaging of fluorescence in over‐1000 nm near‐infrared (OTN‐NIR), which allows in vivo deep imaging, using computed tomography (CT) system. Cylindrical specimens of composite of OTN‐NIR fluorophore, NaGdF₄ co‐doped with Yb³⁺ and Ho³⁺ (ex: 980 nm, em: 1150 nm), were embedded in cubic agar (10.5–12 mm) or in the peritoneal cavity of mice and placed on a rotatable stage. When the fluorescence from inside of the samples was serially captured from multiple angles, the images were disrupted by the reflection and refraction of emitted light on the sample‐air interface. Immersing the sample into water filled in a rectangular bath suppressed the disruption at the interface and successfully reconstructed the position and concentration of OTN‐NIR fluorophores on the cross‐sectional images using a CT technique. This is promising as a novel three‐dimensional imaging technique for OTN‐NIR fluorescent image projections of small animals captured from multiple angles.     

A high‐throughput all‐optical laser‐scanning imaging flow cytometer with biomolecular specificity and subcellular resolution

Image‐based cellular assay advances approaches to dissect complex cellular characteristics through direct visualization of cellular functional structures. However, available technologies face a common challenge, especially when it comes to the unmet need for unraveling population heterogeneity at single‐cell precision: higher imaging resolution (and thus content) comes at the expense of lower throughput, or vice versa. To overcome this challenge, a new type of imaging flow cytometer based upon an all‐optical ultrafast laser‐scanning imaging technique, called free‐space angular‐chirp‐enhanced delay (FACED) is reported. It enables an imaging throughput (>20 000 cells s^?1) 1 to 2 orders of magnitude higher than the camera‐based imaging flow cytometers. It also has 2 critical advantages over optical time‐stretch imaging flow cytometry, which achieves a similar throughput: (1) it is widely compatible to the repertoire of biochemical contrast agents, favoring biomolecular‐specific cellular assay and (2) it enables high‐throughput visualization of functional morphology of individual cells with subcellular resolution. These capabilities enable multiparametric single‐cell image analysis which reveals cellular heterogeneity, for example, in the cell‐death processes demonstrated in this work—the information generally masked in non‐imaging flow cytometry. Therefore, this platform empowers not only efficient large‐scale single‐cell measurements, but also detailed mechanistic analysis of complex cellular processes.      

Inside Front Cover: Slide‐free imaging of hematoxylin‐eosin stained whole‐mount tissues using combined third‐harmonic generation and three‐photon fluorescence microscopy (J. Biophotonics 5/2019)

Intraoperative margin assessment is clinically important, especially for tissue conserving surgery like Mohs micrographic surgery in which minimization of the surgical area is crucial. Instead of the complex frozen pathology protocol, slide‐free histopathological imaging of hematoxylin‐eosin stained whole‐mount skin tissues is demonstrated by using nonlinear microscopy, thus facilitating rapid intraoperative assessment of surgical tissues for future applications. Further details can be found in the article by Chi‐Kuang Sun, Chien‐Ting Kao, Ming‐Liang Wei, et al. ( e201800341 ).

     

Cellular transformations in near‐infrared light‐induced apoptosis in cancer cells revealed by label‐free CARS imaging

Photobiomodulation (PBM) involves light to activate cellular signaling pathways leading to cell proliferation or death. In this work, fluorescence and Coherent anti‐Stokes Raman Scattering (CARS) imaging techniques were applied to assess apoptosis in human cervical cancer cells (HeLa) induced by near infrared (NIR) laser light (808 nm). Using the Caspase 3/7 fluorescent probe to identify apoptotic cells, we found that the pro‐apoptotic effect is significantly dependent of irradiation dose. The highest apoptosis rate was noted for the lower irradiation doses, that is, 0.3 J/cm² (~58%) and 3 J/cm² (~28%). The impact of light doses on proteins/lipids intracellular metabolism and distribution was evaluated using CARS imaging, which revealed apoptosis‐associated reorganization of nuclear proteins and cytoplasmic lipids after irradiation with 0.3 J/cm². Doses of NIR light causing apoptosis (0.3, 3 and 30 J/cm²) induced a gradual increase in the nuclear protein level over time, in contrast to proteins in cells non‐irradiated and irradiated with 10 J/cm². Furthermore, irradiation of the cells with the 0.3 J/cm² dose resulted in lipid droplets (LDs) accumulation, which was apparently caused by an increase in reactive oxygen species (ROS) generation. We suggest that PBM induced apoptosis could be caused by the ability of NIR light to trigger excessive LDs formation which, in turn, induces cellular cytotoxicity.      

Autofluorescence and white light imaging‐guided endoscopic Raman and diffuse reflectance spectroscopy for in vivo nasopharyngeal cancer detection

Nasopharyngeal cancer (NPC) is an endemic with high incidence in Southern China and Southeast Asia countries. Screening for NPC under conventional white light imaging (WLI) nasopharyngoscope examination remains a great clinical challenge due to its poor sensitivity. Here, we developed an integrated 4‐modality endoscopy system combining WLI, autofluorescence imaging (AFI), diffuse reflectance spectroscopy and Raman spectroscopy technologies for in vivo endoscopic cancer detection for the first time. A pilot clinical test of the system for NPC detection was conducted, in which 283 in vivo Raman and diffuse reflectance spectral data sets from 30 NPC patients and 30 healthy subjects were acquired under the guidance of AFI and WLI. Both high diagnostic sensitivity (98.6%) and high specificity (95.1%) for differentiating cancer from normal tissue sites were achieved using this system combined with principal component analysis‐linear discriminant analysis diagnostic algorithm, demonstrating great potential for improving real‐time, in vivo diagnosis of NPC at endoscopy.      

Relationships between serotonergic and cannabinoid system in depressive‐like behavior: a PET study with [11C]‐DASB

Chronic stress represents a major environmental risk factor for mood disorders in vulnerable individuals. The neurobiological mechanisms underlying these disorders involve serotonergic and endocannabinoid systems. In this study, we have investigated the relationships between these two neurochemical systems in emotional control using genetic and imaging tools. CB₁ cannabinoid receptor knockout mice (KO) and wild‐type littermates (WT) were exposed to chronic restraint stress. Depressive‐like symptoms (anhedonia and helplessness) were produced by chronic stress exposure in WT mice. CB₁ KO mice already showed these depressive‐like manifestations in non‐stress conditions and the same phenotype was observed after chronic restraint stress. Chronic stress similarly impaired long‐term memory in both genotypes. In addition, brain levels of serotonin transporter (5‐HTT) were assessed using positron emission tomography. Decreased brain 5‐HTT levels were revealed in CB₁ KO mice under basal conditions, as well as in WT mice after chronic stress. Our results show that chronic restraint stress induced depressive‐like behavioral alterations and brain changes in 5‐HTT levels similarly to those revealed in CB₁ KO mice in non‐stressed conditions. These results underline the relevance of chronic environmental stress on serotonergic and endocannabinoid transmission for the development of depressive symptoms.

     

Low‐photobleaching line‐scanning confocal microscopy using dual inclined beams

Confocal microscopy is an indispensable tool for biological imaging due to its high resolution and optical sectioning capability. However, its slow imaging speed and severe photobleaching have largely prevented further applications. Here, we present dual inclined beam line‐scanning (LS) confocal microscopy. The reduced excitation intensity of our imaging method enabled a 2‐fold longer observation time of fluorescence compared to traditional LS microscopy while maintaining a good sectioning capability and single‐molecule sensitivity. We characterized the performance of our method and applied it to subcellular imaging and three‐dimensional single‐molecule RNA imaging in mammalian cells.