Inside Cover: Noninvasive,high‐speed,near‐infrared imaging of the biomolecular distribution and molecular mechanism of embryonic development in fertilized fish eggs (J. Biophotonics 4/2018)

The biomaterial distribution and its molecular mechanism of embryonic development in Japanese medaka fish were visualized without staining using high‐speed near‐infrared imaging. It was a remarkable achievement to visualize the structures of eyes, lipid bilayer membranes, micelles, and water structural variations at the interface of different substances. Furthermore, insights on lipid metabolism and membrane functions were obtained from the biased distribution of lipoproteins and the presence of unsaturated fatty acids in the egg membrane. Further details can be found in the article by Mika Ishigaki ( e201700115 )

     

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.      

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.      

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.      

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.      

Transmission in near‐infrared optical windows for deep brain imaging

Near‐infrared (NIR) radiation has been employed using one‐ and two‐photon excitation of fluorescence imaging at wavelengths 650–950 nm (optical window I) for deep brain imaging; however, longer wavelengths in NIR have been overlooked due to a lack of suitable NIR‐low band gap semiconductor imaging detectors and/or femtosecond laser sources. This research introduces three new optical windows in NIR and demonstrates their potential for deep brain tissue imaging. The transmittances are measured in rat brain tissue in the second (II, 1,100–1,350 nm), third (III, 1,600–1,870 nm), and fourth (IV, centered at 2,200 nm) NIR optical tissue windows. The relationship between transmission and tissue thickness is measured and compared with the theory. Due to a reduction in scattering and minimal absorption, window III is shown to be the best for deep brain imaging, and windows II and IV show similar but better potential for deep imaging than window I.

     

Activity assessment of root caries lesions with thermal and near‐IR imaging methods

The purpose of this study was to evaluate thermal and near‐infrared (NIR) reflectance imaging methods for the assessment of the activity of root caries lesions. In addition, changes in the lesion structure were monitored with polarization sensitive optical coherence tomography (PS‐OCT). Artificial bovine and natural root caries lesions were imaged with PS‐OCT, and their dehydration rate was measured with thermal and NIR cameras. The lesion activity of the natural root caries samples was also assessed by two clinicians by conventional means according to ICDAS II guidelines. The thickness of the highly mineralized transparent surface layer measured using PS‐OCT increased and the area enclosed by the time‐temperature curve, ΔQ, measured with thermal imaging decreased significantly with longer periods of remineralization in simulated dentin lesions, but the NIR reflectance intensity differences, ΔI, failed to show any significant relationship with the degree of remineralization. The PS‐OCT algorithm for the automated assessment of remineralization successfully detected the highly mineralized surface layer on both natural and simulated lesions. Thermal imaging provided the most accurate diagnosis of root caries lesion activity. These results demonstrate that thermal imaging and PS‐OCT may be ideally suited for the nondestructive root caries lesion activity during a clinical examination.

     

In vitro and in vivo phasor analysis of stoichiometry and pharmacokinetics using short‐lifetime near‐infrared dyes and time‐gated imaging

We introduce a simple new approach for time‐resolved multiplexed analysis of complex systems using near‐infrared (NIR) dyes, applicable to in vitro and in vivo studies. We show that fast and precise in vitro quantification of NIR fluorophores' short (subnanosecond) lifetime and stoichiometry can be done using phasor analysis, a computationally efficient and user‐friendly representation of complex fluorescence intensity decays obtained with pulsed laser excitation and time‐gated camera imaging. We apply this approach to the study of binding equilibria by Förster resonant energy transfer using two different model systems: primary/secondary antibody binding in vitro and ligand/receptor binding in cell cultures. We then extend it to dynamic imaging of the pharmacokinetics of transferrin engagement with the transferrin receptor in live mice, elucidating the kinetics of differential transferrin accumulation in specific organs, straightforwardly differentiating specific from nonspecific binding. Our method, implemented in a freely‐available software, has the advantage of time‐resolved NIR imaging, including better tissue penetration and background‐free imaging, but simplifies and considerably speeds up data processing and interpretation, while remaining quantitative. These advances make this method attractive and of broad applicability for in vitro and in vivo molecular imaging and could be extended to applications as diverse as image‐guided surgery or optical tomography.      

Inside Cover: Cellular transformations in near‐infrared light‐induced apoptosis in cancer cells revealed by label‐free CARS imaging (J. Biophotonics 12/2019)

CARS microscopy was employed to monitor the reorganization of intracellular lipids and proteins, as well as cellular transformations, after irradiation with near infrared (NIR) light. NIR light was shown to induce apoptosis in HeLa cells in vitro in a dose‐dependent manner. The progression of apoptosis assessed with CARS microscopy is apparently associated with the generation of reactive oxygen species followed by an excessive formation of lipid droplets and their peroxidation. Further details can be found in the article by Svitlana M. Levchenko, Andrey N. Kuzmin, Artem Pliss, et al. ( e201900179 )

     

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.      

Assessment of the metabolism and morphology of the porcine cornea,lens and retina by 2‐photon imaging

Two‐photon imaging is a noninvasive imaging technique with increasing importance in the biological and medical fields since it allows intratissue cell imaging with high resolution. We demonstrate the feasibility of using a single 2‐photon instrument to evaluate the cornea, the crystalline lens and the retina based on their autofluorescence (AF). Image acquisition was performed using a custom‐built 2‐photon microscope for 5‐dimensional microscopy with a near infrared broadband sub‐15 femtosecond laser centered at 800 nanometers. Signals were detected using a spectral photomultiplier tube. The spectral ranges for the analysis of each tissue/layer AF were determined based on the spectra of each tissue as well as of pure endogenous fluorophores. The cornea, lens and retina are characterized at multiple depths with subcellular resolution based on their morphology and AF lifetime. Additionally, the AF lifetime of NAD(P)H was used to assess the metabolic activity of the cornea epithelium, endothelium and keratocytes. The feasibility to evaluate the metabolic activity of lens epithelial cells was also demonstrated, which may be used to further investigate the pathogenesis of cataracts. The results illustrate the potential of multimodal multiphoton imaging as a novel ophthalmologic technique as well as its potential as a diagnostic tool.      

K‐distribution three‐dimensional mapping of biological tissues in optical coherence tomography

Probability density function (PDF) analysis with K‐distribution model of optical coherence tomography (OCT) intensity signals has previously yielded a good representation of the average number of scatterers in a coherence volume for microspheres‐in‐water systems, and has shown initial promise for biological tissue characterization. In this work, we extend these previous findings, based on single point M‐mode or two‐dimenstional slice analysis, to full three‐dimensional (3D) imaging maps of the shape parameter α of the K‐distribution PDF. After selecting a suitably sized 3D evaluation window, and verifying methodology in phantoms, the resultant parametric α images obtained in different animal tissues (rat liver and brain) show new contrasting ability not seen in conventional OCT intensity images.      

Trafficking of Acetyl‐C16‐Ceramide‐NBD with Long‐Term Stability and No Cytotoxicity into the Golgi Complex

The Golgi complex plays a prominent role in the modification and sorting of lipids and proteins, and is a highly dynamic organelle that is dispersed and rearranged before and after mitosis. Several reagents including 4‐nitrobenzo‐2‐oxa‐1,3‐diazole‐labeled C6‐ceramide (NBD‐C6‐ceramide, a ceramide having an NBD‐bound C6‐N‐acyl chain) and Golgi‐specific proteins that emit fluorescence are used as Golgi markers. In the present study, we synthesized a new ceramide analog, acetyl‐C16‐ceramide‐NBD (a ceramide having an acetylated C‐1 hydroxyl group, C16‐N‐acyl chain, and NBD‐bound C15‐sphingosine), and showed that it preferentially accumulated in the Golgi complex without cytotoxicity for over 24 h. Pathways for cellular uptake and interorganelle trafficking of acetyl‐C16‐ceramide‐NBD were investigated. Acetyl‐C16‐ceramide‐NBD was transported to the Golgi complex via ceramide transport proteins. In contrast to NBD‐C6‐ceramide, acetyl‐C16‐ceramide‐NBD was resistant to ceramide metabolic enzymes such as sphingomyelin synthase and glucosylceramide synthase. Because of its weaker cytotoxicity and resistance to ceramide metabolic enzymes, the localization of the Golgi complex could be observed in acetyl‐C16‐ceramide‐NBD‐labeled cells before and after mitosis.      

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.     

Label‐free,zeptomole cancer biomarker detection by surface‐enhanced fluorescence on nanoporous gold disk plasmonic nanoparticles

We experimentally demonstrate a label‐free biosensor for the ERBB2 cancer gene DNA target based on the distance‐dependent detection of surface‐enhanced fluorescence (SEF) on nanoporous gold disk (NPGD) plasmonic nanoparticles. We achieve detection of 2.4 zeptomole of DNA target on the NPGD substrate with an upper concentration detection limit of 1 nM. Without the use of molecular spacers, the NPGD substrate as an SEF platform was shown to provide higher net fluorescence for visible and NIR fluorophores compared to glass and non‐porous gold substrates. The enhanced fluorescence signals in patterned nanoporous gold nanoparticles make NPGD a viable material for further reducing detection limits for biomolecular targets used in clinical assays.

With patterned nanoporous gold disk (NPGD) plasmonic nanoparticles, a label‐free biosensor that makes use of distance‐dependent detection of surface‐enhanced fluorescence (SEF) is constructed and tested for zeptomole detection of ERBB2 cancer gene DNA targets.     

Autofluorescence imaging identifies tumor cell‐cycle status on a single‐cell level

The goal of this study is to validate fluorescence intensity and lifetime imaging of metabolic co‐enzymes NAD(P)H and FAD (optical metabolic imaging, or OMI) as a method to quantify cell‐cycle status of tumor cells. Heterogeneity in tumor cell‐cycle status (e. g. proliferation, quiescence, apoptosis) increases drug resistance and tumor recurrence. Cell‐cycle status is closely linked to cellular metabolism. Thus, this study applies cell‐level metabolic imaging to distinguish proliferating, quiescent, and apoptotic populations. Two‐photon microscopy and time‐correlated single photon counting are used to measure optical redox ratio (NAD(P)H fluorescence intensity divided by FAD intensity), NAD(P)H and FAD fluorescence lifetime parameters. Redox ratio, NAD(P)H and FAD lifetime parameters alone exhibit significant differences (p<0.05) between population means. To improve separation between populations, linear combination models derived from partial least squares ‐ discriminant analysis (PLS‐DA) are used to exploit all measurements together. Leave‐one‐out cross validation of the model yielded high classification accuracies (92.4 and 90.1 % for two and three populations, respectively). OMI and PLS‐DA also identifies each sub‐population within heterogeneous samples. These results establish single‐cell analysis with OMI and PLS‐DA as a label‐free method to distinguish cell‐cycle status within intact samples. This approach could be used to incorporate cell‐level tumor heterogeneity in cancer drug development.

     

4‐alkoxyethoxy‐N‐octadecyl‐1,8‐naphthalimide fluorescent sensor for human serum albumin and other major blood proteins: design,synthesis and solvent effect

A series of 4‐alkoxyethoxy‐N‐octadecyl‐1,8‐naphthalimides with intense blue fluorescence were designed and synthesized as polarity and spectrofluorimetric probes for the determination of proteins. In solvents of different polarities, the Stokes shifts of two dyes increased with increasing solvent polarity and fluorescence quantum yields decreased significantly, suggesting that electronic transiting from ground to excited states was π–π^* in character. Dipole moment changes were estimated from solvent‐dependent Stokes shift data using a solvatochromic method based on bulk solvent polarity functions and the microscopic solvent polarity parameter (). These results were generally consistent with semi‐empirical molecular orbital calculations and were found to be quite reliable based on the fact that the correlation of the solvatochromic Stokes shifts with was superior to that obtained using bulk solvent polarity functions. Fluorescence data revealed that the fluorescence quenching of human serum albumin (HSA) by dyes was the result of the formation of a Dye–HSA complex. The method was applied to the determination of total proteins (HSA + immunoglobulins) in human serum samples and results were in good agreement with those reported by the research institute. Copyright © 2012 John Wiley & Sons, Ltd.     

Portable optical‐resolution photoacoustic microscopy for volumetric imaging of multiscale organisms

Photoacoustic microscopy (PAM) provides a fundamentally new tool for a broad range of studies of biological structures and functions. However, the use of PAM has been largely limited to small vertebrates due to the large size/weight and the inconvenience of the equipment. Here, we describe a portable optical‐resolution photoacoustic microscopy (pORPAM) system for 3‐dimensional (3D) imaging of small‐to‐large rodents and humans with a high spatiotemporal resolution and a large field of view. We show extensive applications of pORPAM to multiscale animals including mice and rabbits. In addition, we image the 3D vascular networks of human lips, and demonstrate the feasibility of pORPAM to observe the recovery process of oral ulcer and cancer‐associated capillary loops in human oral cavities. This technology is promising for broad biomedical studies from fundamental biology to clinical diseases.      

Detergent‐dependent separation of postsynaptic density,membrane rafts and other subsynaptic structures from the synaptic plasma membrane of rat forebrain

We systematically investigated the purification process of post‐synaptic density (PSD) and post‐synaptic membrane rafts (PSRs) from the rat forebrain synaptic plasma membranes by examining the components and the structures of the materials obtained after the treatment of synaptic plasma membranes with TX‐100, n‐octyl β‐d ‐glucoside (OG) or 3‐([3‐cholamidopropyl]dimethylammonio)‐2‐hydroxy‐1‐propanesulfonate (CHAPSO). These three detergents exhibited distinct separation profiles for the synaptic subdomains. Type I and type II PSD proteins displayed mutually exclusive distribution. After TX‐100 treatment, type I PSD was recovered in two fractions: a pellet and an insoluble fraction 8, which contained partially broken PSD‐PSR complexes. Conventional PSD was suggested to be a mixture of these two PSD pools and did not contain type II PSD. An association of type I PSD with PSRs was identified in the TX‐100 treatment, and those with type II PSD in the OG and CHAPSO treatments. An association of GABA receptors with gephyrin was easily dissociated. OG at a high concentration solubilized the type I PSD proteins. CHAPSO treatment resulted in a variety of distinct fractions, which contained certain novel structures. Two different pools of GluA, either PSD or possibly raft‐associated, were identified in the OG and CHAPSO treatments. These results are useful in advancing our understanding of the structural organization of synapses at the molecular level.

     

A possible Fourier transform infrared‐based plasma fingerprint of angiotensin‐converting enzyme inhibitor‐induced reversal of endothelial dysfunction in diabetic mice

Angiotensin‐converting enzyme inhibitors (ACE‐I) display vasoprotective activity and represent the cornerstone in the treatment of cardiovascular diseases. In this study, we tested whether Fourier transform infrared (FTIR)‐based analysis of blood plasma is sensitive to detect vasoprotective effects of treatment with perindopril including reversal of endothelial dysfunction in diabetes. For this purpose, plasma samples were collected from untreated db/db mice, db/db mice treated with 2 or 10 mg/kg perindopril and db+ mice. The effect of perindopril on endothelial function was examined in ex vivo aortic rings; 10 mg/kg but not 2 mg/kg of perindopril reversed endothelial dysfunction. In plasma of db/db mice, the balance between conformations of plasma proteins was noted, and treatment with perindopril at a high dose but not at a low dose reversed this effect. This was revealed by amide II/amide I ratio attributed to increased β‐sheet formation. Spectral markers at 3010, 1520/1238 cm^?1, representative for unsaturation degree of lipids and phosphorylation of tyrosine, respectively, were also affected by perindopril treatment. In conclusion, although metabolic abnormalities associated with type 2 diabetes mellitus such as hypertriglyceridemia and hyperglycemia strongly affected spectral FTIR profile of diabetic plasma, we identified FTIR features that seem to be associated with the vasoprotective activity of ACE‐I.