Search

Emerging studies have shown that lipid metabolism plays an important role in aging. High resolution in situ imaging of lipid metabolic dynamics inside cells and tissues affords a novel and potent approach for understanding many biological processes such as aging. Here we established a new optical imaging platform that combines D2O-probed stimulated Raman scattering (DO-SRS) imaging microscopy and a Drosophila model to directly visualize metabolic activities in situ during aging. The sub-cellular spatial distribution of de novo lipogenesis in the fat body was quantitatively imaged and examined. We discovered a dramatic decrease in lipid turnover in 35-day-old flies. Decreases in protein turnover occurred earlier than lipids (25-day vs. 35-day), and there are many proteins localized on the cell and lipid droplet membrane. This suggests that protein metabolism may act as a prerequisite for lipid metabolism during aging. This alteration of maintenance of protein turnover indicates disrupted lipid metabolism. We further found a significantly higher lipid turnover rate in large LDs, indicating more active metabolism in large LDs, suggesting that large and small LDs play different roles in metabolism to maintain cellular homeostasis. This is the first study that directly visualizes spatiotemporal alterations of lipid (and protein) metabolism in Drosophila during the aging process. Our study not only demonstrates a new imaging platform for studying lipid metabolism, but also unravels the important interconnections between lipid metabolism and aging.

Understanding metabolism is indispensable to unraveling the mechanistic basis of many physiological and pathological processes. However, in situ metabolic imaging tools are still lacking. Herein we introduce a framework for mid-infrared (MIR) metabolic imaging by coupling the emerging high-information-throughput MIR microscopy with specifically designed IR-active vibrational probes. Three categories of small vibrational tags including azide bond, 13C-edited carbonyl bond and deuterium-labeled probes are presented to interrogate various metabolic activities in cells, small organisms and mice. Two MIR imaging platforms are implemented including broadband Fourier transform infrared (FTIR) microscopy and discrete frequency infrared (DFIR) microscopy with a newly incorporated spectral region (2000–2300 cm−1). Our technique is uniquely suited for metabolic imaging with high-information-throughput. In particular, we performed single-cell metabolic profiling including heterogeneity characterization, and large-area metabolic imaging at tissue/organ level with rich spectral information., Editor’s summary Small vibrational tags (azide, 13C-edited carbonyl, and deuterium-labeled probes) were introduced as metabolic probes for mid-infrared imaging. The tags allow unprecedented in situ visualization of metabolism in cells and animals with high information-throughput.

Defining the relationship between vascular development and the expression of hypoxia-inducible factors (Hifs) and vascular endothelial growth factor (Vegf) in the auditory brainstem is important to understand how tissue hypoxia caused by oxygen shortage contributes to sensory deficits in neonates. In this study, we used histology, molecular labeling, confocal microscopy and 3D image processing methods to test the hypothesis that significant maturation of the vascular bed in the medial nucleus of the trapezoid body (MNTB) occurs during the postnatal period that precedes hearing onset. Isolectin-B4 histochemistry experiments suggested that the MNTB vasculature becomes more elaborate between P5 and P10. When combined with a cell proliferation marker and immunohistochemistry, we found that vascular growth coincides with a switch in the localization of proliferating cells to perivascular locations, and an increase in the density of microglia within the MNTB. Furthermore, microglia were identified as perivascular cells with proliferative activity during the period of vascular maturation. Lastly, combined in situ hybridization and immunohistochemistry experiments showed distinct profiles of Hif1a and Vegf mRNA localization in microglia, astrocytes and MNTB principal neurons. These results suggest that different cells of the neuro-glio-vascular unit are likely targets of hypoxic insult in the auditory brainstem of neonate rats.

Oxaliplatin is one of the most commonly used chemotherapeutic agents in the treatment of various cancers, including gastric cancer. It has, however, a narrow therapeutic index due to its toxicity and the occurrence of drug resistance. Therefore, there is a pressing need to develop novel therapies to potentiate the efficacy and reduce the toxicity of oxaliplatin. Piperlongumine (PL), an alkaloid isolated from Piper longum L., has recently been identified as a potent agent against cancer cells in vitro and in vivo. In the present study, we investigated whether PL can potentiate the antitumor effect of oxaliplatin in gastric cancer cells. Cellular apoptosis and ROS levels were analyzed by flow cytometry. Thioredoxin reductase 1 (TrxR1) activity in gastric cancer cells or tumor tissues was determined using an endpoint insulin reduction assay. Western blotting was used to analyze the expression levels of the indicated proteins. Nude mice xenograft models were used to test the effects of PL and oxaliplatin combinations on gastric cancer cell growth in vivo. We found that PL significantly enhanced oxaliplatin-induced growth inhibition in both gastric and colon cancer cells. Moreover, we found that PL potentiated the antitumor effect of oxaliplatin by inhibiting TrxR1 activity. PL combined with oxaliplatin markedly suppressed the activity of TrxR1, resulting in the accumulation of ROS and, thereby, DNA damage induction and p38 and JNK signaling pathway activation. Pretreatment with antioxidant N-acetyl-L-cysteine (NAC) significantly abrogated the combined treatment-induced ROS generation, DNA damage and apoptosis. Importantly, we found that activation of the p38 and JNK signaling pathways prompted by PL and oxaliplatin was also reversed by NAC pretreatment. In vivo, we found that PL combined with oxaliplatin significantly suppressed tumor growth in a gastric cancer xenograft model, and effectively reduced the activity of TrxR1 in tumor tissues. Remarkably, we found that PL attenuated body weight loss evoked by oxaliplatin treatment. Our data support a synergistic effect of PL and oxaliplatin and suggest that application of its combination may be more effective for the treatment of gastric cancer than oxaliplatin alone.

Breast cancer is a diverse disease rife with numerous subtypes with material impact on prognoses. Current methods may lack accuracy or be cost and time prohibitive, but optical techniques such as Isotope Probed Stimulated Raman Scattering microscopy (ip-SRS), and two photon excitation fluorescence (TPEF) microscopy can reveal spatial biomolecular information useful in distinguishing cell subtypes and phenotypic states rapidly and accurately. In the present study, we used heavy water and L-methionine to probe the enzymatic incorporation during scavenging and de novo biosynthesis of macromolecules in MCF10A, MCF7, and MDA-MB-231 breast cancer cells with spontaneous Raman spectro-microscopy and SRS microscopy, as well as their effects on cellular respiration and organelle health by imaging the NADH and flavin pools and labeled organelles with TPEF. This will enhance diagnostic efficacy and illustrate specific biochemical effects of manipulated nutrition and targeted therapies.

Early diagnosis of Triple Negative Breast Cancer (TNBC) is essential to implementing early, life-saving treatment before the development of metastases. Traditional methods for detecting TNBC is difficult, being both tedious and vulnerable to false positive results. Here we combine optical imaging techniques—deuterium probed resonance Raman spectroscopy (RRS) and multiphoton fluorescence (MPF) to detect TNBC metabolism in early stage. These hallmarks such as glucose and lipid metabolism are revealed through chemical bond vibrational modes with RRS and morphological changes with MPF at subcellular scale.

Brain is a complex organ displaying cellular structural and functional heterogeneity. Understanding the dynamics of metabolism in brain with cellular resolution is essential to unraveling the mechanistic basis of many neuronal activities. Traditional imaging methods cannot provide metabolic activity information with high resolution in situ. We developed a new method that combines bioorthogonal labeling and Raman Scattering microscopy to visualize metabolic dynamics in brains. For example, the incorporation of deuterated glucose, or heavy water (D2O) into biomolecules will generate new carbon-deuterium (C-D) bonds in protein, lipids and nucleic acids in brain. We obtained new insights through high resolution imaging of whole brain, such as new protein and lipids synthesis in different brain regions during the developmental and aging processes. We demonstrate that this is an efficient methods for visualizing metabolic activities in brain.

Direct imaging of metabolism in cells or multicellular organisms is important for understanding many biological processes. Raman scattering (RS) microscopy, particularly, coherent Raman scattering (CRS) such as coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS), has emerged as a powerful platform for cellular imaging due to its high chemical selectivity, sensitivity, and imaging speed. RS microscopy has been extensively used for the identification of subcellular structures, metabolic observation, and phenotypic characterization. Conjugating RS modalities with other techniques such as fluorescence or infrared (IR) spectroscopy, flow cytometry, and RNA-sequencing can further extend the applications of RS imaging in microbiology, system biology, neurology, tumor biology and more. Here we overview RS modalities and techniques for mammalian cell and tissue imaging, with a focus on the advances and applications of CARS and SRS microscopy, for a better understanding of the metabolism and dynamics of lipids, protein, glucose, and nucleic acids in mammalian cells and tissues. This article is categorized under: Laboratory Methods and Technologies > Imaging Biological Mechanisms > Metabolism Analytical and Computational Methods > Analytical Methods.

A two-color vibrational imaging technique for simultaneously mapping glucose uptake and incorporation activity inside single living cells is reported. Heterogeneous patterns of glucose metabolism are directly visualized from the ratiometric two-color images for various cell types, cells undergoing epithelia-to-mesenchymal transitions and live mouse brain tissues. The two-color imaging of glucose metabolism here demonstrates the development of multi-functional vibrational probes for multicolor imaging of cellular metabolism.

Significance Membranes can adopt distinct phases. The endoplasmic reticulum (ER) is the largest membrane system inside cells and also harbors the richest metabolic activity including lipid synthesis. Unlike plasma membrane where separated “lipid raft” domains have been predicted and observed, ER membrane is thought to be uniformly fluidic. However, such understanding is based on biophysical studies of model membrane under thermodynamic equilibrium. It remains unclear whether and how lipid synthesis activity perturbs the equilibrium and promotes phase segregation in ER membrane. Herein, we utilized coherent Raman imaging technique to track lipid synthesis and surprisingly revealed solid-like domains emerging from liquid ER membrane. Interestingly, this phenomenon can be tuned by the incoming nutrient source, demonstrating the susceptibility of ER membrane to nonequilibrium modulation., Membrane phase behavior has been well characterized in model membranes in vitro under thermodynamic equilibrium state. However, the widely observed differences between biological membranes and their in vitro counterparts are placing more emphasis on nonequilibrium factors, including influx and efflux of lipid molecules. The endoplasmic reticulum (ER) is the largest cellular membrane system and also the most metabolically active organelle responsible for lipid synthesis. However, how the nonequilibrium metabolic activity modulates ER membrane phase has not been investigated. Here, we studied the phase behavior of functional ER in the context of lipid metabolism. Utilizing advanced vibrational imaging technique, that is, stimulated Raman scattering microscopy, we discovered that metabolism of palmitate, a prevalent saturated fatty acid (SFA), could drive solid-like domain separation from the presumably uniformly fluidic ER membrane, a previously unknown phenomenon. The potential of various fatty acids to induce solid phase can be predicted by the transition temperatures of their major metabolites. Interplay between saturated and unsaturated fatty acids is also observed. Hence, our study sheds light on cellular membrane biophysics by underscoring the nonequilibrium metabolic status of living cell.

Glioma is one of the most refractory types of brain tumor. Accurate tumor boundary identification and complete resection of the tumor are essential for glioma removal during brain surgery. We present a method based on visible resonance Raman (VRR) spectroscopy to identify glioma margins and grades. A set of diagnostic spectral biomarkers features are presented based on tissue composition changes revealed by VRR. The Raman spectra include molecular vibrational fingerprints of carotenoids, tryptophan, amide I/II/III, proteins, and lipids. These basic in situ spectral biomarkers are used to identify the tissue from the interface between brain cancer and normal tissue and to evaluate glioma grades. The VRR spectra are also analyzed using principal component analysis for dimension reduction and feature detection and support vector machine for classification. The cross-validated sensitivity, specificity, and accuracy are found to be 100%, 96.3%, and 99.6% to distinguish glioma tissues from normal brain tissues, respectively. The area under the receiver operating characteristic curve for the classification is about 1.0. The accuracies to distinguish normal, low grade (grades I and II), and high grade (grades III and IV) gliomas are found to be 96.3%, 53.7%, and 84.1% for the three groups, respectively, along with a total accuracy of 75.1%. A set of criteria for differentiating normal human brain tissues from normal control tissues is proposed and used to identify brain cancer margins, yielding a diagnostic sensitivity of 100% and specificity of 71%. Our study demonstrates the potential of VRR as a label-free optical molecular histopathology method used for in situ boundary line judgment for brain surgery in the margins.

Cells and tissues often display pronounced spatial and dynamical metabolic heterogeneity. Prevalent glucose-imaging techniques report glucose uptake or catabolism activity, yet do not trace the functional utilization of glucose-derived anabolic products. Here, we report a microscopy technique for the optical imaging, via the spectral tracing of deuterium (referred to as STRIDE), of diverse macromolecules derived from glucose. Based on stimulated-Raman-scattering imaging, STRIDE visualizes the metabolic dynamics of newly synthesized macromolecules, such as DNA, protein, lipids and glycogen, via the enrichment and distinct spectra of carbon–deuterium bonds transferred from the deuterated glucose precursor. STRIDE can also use spectral differences derived from different glucose isotopologues to visualize temporally separated glucose populations in a pulse–chase manner. We also show that STRIDE can be used to image glucose metabolism in many mouse tissues, including tumours, the brain, the intestine and the liver, at a detection limit of 10 mM of carbon–deuterium bonds. STRIDE provides a high-resolution and chemically informative assessment of glucose anabolic utilization.

Understanding the dynamics of metabolism in a multicellular organism is essential to unraveling the mechanistic basis of many biological processes. It is the synthesis, transformation and degradation of biomolecules (the definition of metabolism) that carry out the genetic blueprint, which cannot be imaged in vivo by using traditional methods. In the present work, we developed a new method that combines D2O probing and Stimulated Raman Scattering microscopy (DO-SRS) to visualize metabolic dynamics in live animals. The enzymatic incorporation of D2O-derived deuterium (D) into biomolecules will generate carbon-deuterium (C-D) bonds in macromolecules. Within the broad vibrational spectra of C-D bonds, we discover lipid-, protein-, and DNA-specific Raman shifts and develop spectral unmixing methods to obtain C-D signals with macromolecular selectivity. We obtained new biological insights in several studies such as the spatial dependence of lipogenic activities of sebaceous glands, specific myelination timing of the developing mouse brain, differential protein and lipid metabolism in germline development of C. elegans as well as its aging process, the spatial constrain for the distribution of newly synthesized yolk proteins in aged C. elegans, the prevalence of protein biosynthesis and the lack of lipogenesis in zebrafish embryos, and intratumoral metabolic heterogeneity. In summary, we demonstrated that our current DO-SRS method is better than other deuterium-labeled carbon substrate in monitoring and imaging metabolic activities. This technique can track specifically de novo lipogenesis, image in vivo protein biosynthesis without tissue bias, and can simultaneously image spatial temporal dynamics of lipid and protein.

Metabolic dynamics is essential to unraveling the mechanistic basis of many biological processes, and cannot be imaged in vivo by using traditional methods. Here we developed a new method that combines D2O probing and SRS (DO-SRS) to visualize metabolic dynamics in live animals. The enzymatic incorporation of D2O-derived deuterium (D) into biomolecules will generate carbon-deuterium (C-D) bonds in macromolecules. We developed spectral unmixing methods to obtain C-D signals with macromolecular selectivity, and applied DO-SRS to reveal metabolic dynamics in C. elegans, zebrafish, and mice, to study developmental biology, aging, and tissue homeostasis, and to visualize tumor boundaries and metabolic heterogeneity.

Direct visualization of metabolic dynamics in living animals with high spatial and temporal resolution is essential to understanding many biological processes. Here we introduce a platform that combines deuterium oxide (D2O) probing with stimulated Raman scattering (DO-SRS) microscopy to image in situ metabolic activities. Enzymatic incorporation of D2O-derived deuterium into macromolecules generates carbon–deuterium (C–D) bonds, which track biosynthesis in tissues and can be imaged by SRS in situ. Within the broad vibrational spectra of C–D bonds, we discover lipid-, protein-, and DNA-specific Raman shifts and develop spectral unmixing methods to obtain C–D signals with macromolecular selectivity. DO-SRS microscopy enables us to probe de novo lipogenesis in animals, image protein biosynthesis without tissue bias, and simultaneously visualize lipid and protein metabolism and reveal their different dynamics. DO-SRS microscopy, being noninvasive, universally applicable, and cost-effective, can be adapted to a broad range of biological systems to study development, tissue homeostasis, aging, and tumor heterogeneity., Non-destructive methods to image metabolism in situ in living tissues are limited. Here the authors combine deuterium oxide probing and stimulated Raman scattering microscopy to image lipid metabolic dynamics and protein synthesis in cells and in vivo in mice, C. elegans, and zebrafish.

Resonance Raman and fluorescence spectroscopy were used to assess increased kynurenine pathway activity in brain samples from Alzheimer’s patients and age-matched controls. Increased activity was seen in areas of the brain involved in Alzheimer’s disease.

The goal of the research is to determine the prognostic molecular pathological changes in components and composition, for human brain glioma gradings in comparison with normal tissues in three-dimensional Raman imaging profiles by visible Resonance Raman (VRR) imaging. VRR images from twenty-five specimens including three healthy tissues, one normal control, and twenty-one glioma tissues of grades II, II-III and III-IV with histology examination were measured and investigated using WITec300R confocal micro Raman imaging system with laser excitation of 532nm. Two-dimensional RR spectral mappings performed in 20μm x 20μm generated 400 images which integrated the intensity of the specific biochemical bonds as the third dimension. The three-dimension (3D) map demonstrated the spatial distributions of three selected sets of RR spectra of molecular biomarkers, and revealed significant differences in the spectra between normal and glioma tissues of different grades due to the composition changes in key molimageecules. These RR molecular spectral fingerprints have displayed: a clear enhancement of RR vibrational modes at 1129-1131cm-1 and 2934cm-1 which are supposed to be arising from lipoproteins; evident decreased RR vibrational modes at 1442cm-1 and 2854cm-1 which are from saturated fatty acids bonds in all-grades of glioma brain tissues compared with normal tissues; and the enhanced RR spectral modes of 1129 cm-1 and 2938cm-1 which suggest contribution from lactate. These findings may provide a novel proof for anaerobic glycolysis metabolic process in brain glioma cancer tissues that has been explained by Warburg effects.

Under stress conditions, pro-inflammatory cytokines, such as tumor necrosis factor-alpha, interleukin-1 beta, interleukin 6 and interferon gamma are released. It is known that these cytokines stimulate indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO), which increase tryptophan metabolism through the kynurenine pathway, and that this can cause increased production of neurotoxic compounds. Brain tissues from Alzheimer’s disease patients and agematched controls were investigated using label-free fluorescence spectroscopy. Tryptophan (exc. 280/ em. 340 nm) and its metabolites (N-formyl-L-kynurenine (exc. 325/em. 434 nm), kynurenine (exc. 365/em. 480 nm) and kynurenic acid (exc. 330/em. 390 nm)) have distinct spectral profiles. Preliminary results show a difference in the optical signatures in three important areas of the brain (hippocampus, BA 9, BA 17) between patients with Alzheimer’s disease and agedmatched controls (normal), and a marked relative increase in tryptophan in the Alzheimer’s patients. Thus determinations of tryptophan to tryptophan metabolite ratios could potentially be used to measure IDO and TDO activity and the degree of inflammation in the brain. This label-free optical technique may be useful in the study of Alzheimer’s and other neurodegenerative diseases.

Brain samples from Alzheimer's patients and aged-matched controls were studied using label-free fluorescence. Increased tryptophan metabolite to tryptophan ratios were seen in the hippocampus and Brodmann's area 9 in Alzheimer's patients as compared to normal.

VRR spectroscopy was used for BCC and normal skin tissues with 532nm excitation. The spectra showed significant changes in collagen, carotenoids and lipids. These enhanced fingerprints demonstrate a potential use as label-free pathology method.

In this study, label-free fluorescence spectroscopy was used for the first time to determine spectral profiles of tryptophan, reduced nicotinamide adenine dinucleotide (NADH), and flavin denine dinucleotide (FAD) in fresh brain samples of a mouse model of Alzheimer’s disease (AD). Our results showed that the emission spectral profile levels of tryptophan and NADH were higher in AD samples than normal samples. The intensity ratio of tryptophan to NADH and the change rate of fluorescence intensity with respect to wavelength also increased in AD brain. These results yield an optical method for detecting early stage of AD by comparing spectral profiles of biomolecules.

Time resolved spectroscopic measurements with single-photon and multi-photon excitation of native molecules were performed ex vivo on brain tissues from an Alzheimer’s disease (AD) and a wild type (WT) mouse model using a streak camera. The fluorescence decay times of native NADH and FAD show a longer relaxation time in AD than in WT tissue, suggesting less non-radiative processes in AD. The longer emission time of AD may be attributed to the coupling of the key native building block molecules to the amyloid-tau and/or to the caging of the native fluorophores by the deposition of amyloid-beta or tau plaques and neurofibrillary tangles that affect the local non-radiative interactions.

To test the hypothesis that vascular endothelial growth factor (VEGF) can transiently increase the blood-brain barrier permeability, P, as for peripheral microvessels and that the elevation of 3,5-cyclic monophosphate (cAMP) levels can inhibit the VEGF-induced acute hyperpermeability, we employed multiphoton microscopy to quantify the cerebral microvessel permeability P to various-sized solutes under VEGF and cAMP treatments. The cerebral microcirculation was observed through a section of frontoparietal bone thinned with a microgrinder. Fluorescein (MW 376Da), fluorescein isothioyanate-dextran-20k (FITC-Dex-20k), FITC-Dex-70k, or Alexa Fluor 488-IgG in 1% bovine serum albumin mammalian Ringer's solution was injected into the cerebral circulation via the ipsilateral carotid artery with a syringe pump. Simultaneously, temporal images were collected from the brain parenchyma ∼100-200 μm below the pia mater. P was determined from the rate of tissue solute accumulation around individual microvessels. Exposure to 1 nM VEGF transiently increased P to 2.2, 10.5, 9.8, and 12.8 times control values, for fluorescein, Dex-20k, Dex-70k, and IgG, respectively, within 30 sec, and all returned to control levels within 2 min. After 20 min of pretreatment with 2 mM of the cAMP analog 8-bromo-cAMP, the initial increase by 1 nM VEGF was completely abolished in P of all solutes. The response pattern of P to VEGF and cAMP and the ratios of the peak to control values for rat cerebral microvessels are similar to those for rat mesenteric (peripheral) microvessels, except that the ratios are higher in P of cerebral microvessels for the intermediate and large solutes. These results imply a new approach for delivering large therapeutic agents to the brain.

Atherosclerosis is characterized by the growth of fibrous plaques due to the retention of cholesterol and lipids within the artery wall, which can lead to vessel occlusion and cardiac events. One way to evaluate arterial disease is to quantify the amount of lipid present in these plaques, since a higher disease burden is characterized by a higher concentration of lipid. Although therapeutic stimulation of reverse cholesterol transport to reduce cholesterol deposits in plaque has not produced significant results, this may be due to current image analysis methods which use averaging techniques to calculate the total amount of lipid in the plaque without regard to spatial distribution, thereby discarding information that may have significance in marking response to therapy. Here we use Directional Fourier Spatial Frequency (DFSF) analysis to generate a characteristic spatial frequency spectrum for atherosclerotic plaques from C57 Black 6 mice both treated and untreated with a cholesterol scavenging nanoparticle. We then use the Cauchy product of these spectra to classify the images with a support vector machine (SVM). Our results indicate that treated plaque can be distinguished from untreated plaque using this method, where no difference is seen using the spatial averaging method. This work has the potential to increase the effectiveness of current in-vivo methods of plaque detection that also use averaging methods, such as laser speckle imaging and Raman spectroscopy.

The blood–brain barrier (BBB) is a unique structure between the cerebral blood circulation and the delicate neural environment that is important in regulating the movement of molecules and ions involved in brain development and function. However, little is known about the physiological permeability of molecules and ions across the BBB during brain development. In this study we applied an innovative approach to examine the development of BBB properties quantitatively. Two-photon microscopy was employed to measure BBB permeability in real time in vivo. Vascular growth and specific interactions between astrocyte end feet and microvessels were studied by using a combination of IB4 histochemistry, immunohistochemistry, confocal microscopy and 3D analysis.

Resonance Raman (RR) spectroscopic technique has a high potential for label-free and in-situ detection of biomedical lesions in vivo. This study evaluates the ability of RR spectroscopy method as an optical histopathology tool to detect the atherosclerotic plaque states of abdominal aorta in vitro. This part demonstrates the RR spectral molecular fingerprint features from different sites of the atherosclerotic abdominal aortic wall tissues. Total 57 sites of five pieces aortic samples in intimal and adventitial wall from an autopsy specimen were examined using confocal micro Raman system of WITec 300R with excitation wavelength of 532nm. The preliminary RR spectral biomarkers of molecular fingerprints indicated that typical calcified atherosclerotic plaque (RR peak at 964cm-1) tissue; fibrolipid plaque (RR peaks at 1007, 1161, 1517 and 2888cm-1) tissue, lipid pool with the fatty precipitation cholesterol) with collagen type I (RR peaks at 864, 1452, 1658, 2888 and 2948cm-1) in the soft tissue were observed and investigated.

The terahertz (THz) absorption and index of refraction of brain tissues from a mouse model of Alzheimer’s disease (AD) and a control wild-type (normal) mouse were compared using THz time-domain spectroscopy (THz-TDS). Three dominating absorption peaks associated to torsional–vibrational modes were observed in AD tissue, at about 1.44, 1.8, and 2.114 THz, closer to the peaks of free tryptophan molecules than in normal tissue. A possible reason is that there is more free tryptophan in AD brain tissue, while in normal brain tissue more tryptophan is attached to other molecules. Our study suggests that THz-absorption modes may be used as an AD biomarker fingerprint in brain, and that THz-TDS is a promising technique for early diagnosis of AD.

The accurate identification of the human brain tumor boundary and the complete resection of the tumor are two essential factors for the removal of the glioma tumor in brain surgery. We present a visible resonance Raman (VRR) spectroscopy technique for differentiating the brain tumor margin and glioma grading. Eighty-seven VRR spectra from twenty-one human brain specimens of four types of brain tissues, including the control, glioma grade II, III, and IV tissues, were observed. This study focuses on observing the characteristics of new biomarkers and their changes in the four types of brain tissue. We found that two new RR peaks at 1129 cm−1 and 1338 cm−1 associated with molecular vibrational bonds in four types of brain tissues are significantly different in peak intensities of VRR spectra. These two resonance enhanced peaks may arise from lactic acid/phosphatidic acid and adenosine triphosphate (ATP)/nicotinamide adenine dinucleotide, respectively. We found that lactic acid and ATP concentrations vary with glioma gratings. The higher the grade of malignancy, the more the increase in lactic acid and ATP concentrations. These two RR peaks may be considered as new molecular biomarkers and used to evaluate glioma grades and identify the margin of gliomas from the control tissues. The metabolic process of lactic acid and ATP in glioma cells based on the VRR spectral changes may reveal the Warburg hypothesis.

Proteins are one of the major components of biological systems, and understanding their metabolism is critical to study various biochemical processes in living systems. Despite extensive efforts to study protein metabolism such as autoradiography, mass spectrometry, and fluorescence microscopy, visualizing the spatial distribution of overall protein metabolism in mammals at subcellular resolution is still challenging. A recent study from our group reported imaging newly synthesized proteins in cultured mammalian cells, tissues, or even in mice using stimulated Raman scattering (SRS) microscopy coupled with metabolic labeling of deuterated amino acids (dAA). However, our previous method of dAA administration via drinking water, albeit convenient, is insufficient for in vivo studies. This is due to poor labeling efficiency and limited access to many important organs such as the brain, pancreas, or tumor. In this study, we have significantly improved and optimized the in vivo administration method by intra-carotid arterial injection of dAA in mice and obtained imaging contrast of protein metabolic activity in many more organs and tissues, such as cerebral and cerebellar cortex and hippocampal regions in the mouse brain. We also imaged newly formed proteins in the choroid plexus and pancreas at different time points, illustrating the metabolic dynamics of proteins in these important secretory organs. In addition, we visualized the metabolic heterogeneity of protein synthesis in colon tumor xenografts, which can be used to distinguish tumor and normal tissues. In summary, this combination of a new dAA administration technique and SRS imaging platform demonstrates an effective tool for the in vivo study of complex protein metabolism in mammals, in both physiological and pathological states.

Ni 80 Fe 20 /SiO 2 /Cu composite wires of Cu core 60 μm in diameter and coated with layers of SiO 2 and Ni 80 Fe 20 were prepared by RF magnetron sputtering. The influences of the insulator layer thickness, the measurement mode and the magnitude of the driving current on the giant magneto-impedance (GMI) effect were investigated. The results showed that there was an optimum thickness of the insulator layer and the driving current can influence the shape of the MI curve. Resonance enhancement of the GMI was found in the new measurement mode. The results are discussed by taking account of the electromagnetic interactions.

Increasing the depth to image inside tissue is critical in biomedicine. Two-photon (2P) excitation of the second singlet (S 2 ) state of a group of fluorescent agents with near infrared emission, Chlorophyll a (Chl a ) and Indocyanine green (ICG), is used to extend the optical imaging regime of 2PM into “tissue optical window” for deep tissue penetration. The fast nonradiative from S 2 to S 1 yields both emission and absorption wavelengths in the therapeutic window. The salient feature is to place both the 2P excitation and emission wavelengths of the imaging agents falling into the “tissue optical window”. As a first step to achieve deeper optical imaging, Chl a and ICG are investigated and demonstrated as imaging agents for 2P S 2 excitation microscope image.

Two-photon (2P) excitation of the second singlet (S₂) state was studied to achieve deep optical microscopic imaging in brain tissue when both the excitation (800 nm) and emission (685 nm) wavelengths lie in the "tissue optical window" (650 to 950 nm). S₂ state technique was used to investigate chlorophyll α (Chl α) fluorescence inside a spinach leaf under a thick layer of freshly sliced rat brain tissue in combination with 2P microscopic imaging. Strong emission at the peak wavelength of 685 nm under the 2P S₂ state of Chl α enabled the imaging depth up to 450 μm through rat brain tissue.

Development of an optimal systemic drug delivery strategy to the brain will require noninvasive or minimally invasive methods to quantify the permeability of the cerebral microvessel wall or blood-brain barrier (BBB) to various therapeutic agents and to measure their transport in the brain tissue. To address this problem, we used laser-scanning multiphoton microscopy to determine BBB permeability to solutes (P) and effective solute diffusion coefficients (Deff) in rat brain tissue 100‐250lm below the pia mater. The cerebral microcirculation was observed through a section of frontoparietal bone thinned with a microgrinder. Sodium fluorescein, fluorescein isothiocyanate (FITC)-dextrans, or Alexa Fluor 488-immunoglobulin G (IgG) in 1% bovine serum albumin (BSA) mammalian Ringer’s solution was injected into the cerebral circulation via the ipsilateral carotid artery by a syringe pump at a constant rate of � 3ml/min. P and Deff were determined from the rate of tissue solute accumulation and the radial concentration gradient around individual microvessels in the brain tissue. The mean apparent permeability P values for sodium fluorescein (molecular weight (MW) 376 Da), dextran-4k, -20k, -40k, -70k, and IgG (MW � 160kDa) were 14.6, 6.2, 1.8, 1.4, 1.3, and 0.54 � 10 � 7 cm/s, respectively. These P values were not significantly different from those of rat pial microvessels for the samesized solutes (Yuan et al., 2009, “Non-Invasive Measurement of Solute Permeability in Cerebral Microvessels of the Rat,” Microvasc. Res., 77(2), pp. 166‐73), except for the small solute sodium fluorescein, suggesting that pial microvessels can be a good model for studying BBB transport of relatively large solutes. The mean Deff values were 33.2, 4.4, 1.3, 0.89, 0.59, and 0.47 � 10 � 7 cm 2 /s, respectively, for sodium fluorescein, dextran4k, -20k, -40k, -70k, and IgG. The corresponding mean ratio of Deff to the free diffusion coefficient Dfree ,D eff/Dfree, were 0.46, 0.19, 0.12, 0.12, 0.11, and 0.11 for these solutes. While there is a significant difference in Deff/Dfree between small (e.g., sodium fluorescein) and larger solutes, there is no significant difference in Deff/Dfree between solutes with molecular weights from 20,000 to 160,000 Da, suggesting that the relative resistance of the brain tissue to macromolecular solutes is similar over a wide size range. The quantitative transport parameters measured from this study can be used to develop better strategies for brain drug delivery. [DOI: 10.1115/1.4025892]

Time-resolved photoluminescence is used to determine carrier recombination through radiative and nonradiative processes in zinc hydroxide Zn(OH)2 and its porous composites with graphite oxide (GO). The decay times, measured by a streak camera, are found to be larger for zinc hydroxide (∼1215±156 ps) than its composites (∼976±81 ps for ZnGO-2 and 742±59 ps for ZnGO-5), but no significant changes in rise times (from 4.0 to 5.0 ps) are recorded. The dominant mechanism for the radiative process is attributed to free carrier recombination, while microporous networks present in these materials are found to be pathways for the nonradiative recombination process via multiphonon emission.

New photovoltaic solar cells based on micro/meso porous Zinc(hydr)oxide with 2% graphite oxide (ZnGO-2) and TiO2 s sensitized by quantum dot and ICG dye are reported. The efficiency of energy conversion has reached up to ~ 6.84%.

Lingyan Shi,1 Paolo Palacio-Mancheno,1 Joseph Badami,2 Da Wi Shin,1 Min Zeng,1 Luis Cardoso,1 Raymond Tu,2 Bingmei M Fu11Department of Biomedical Engineering, 2Department of Chemical Engineering, The City College of the City University of New York, New York, NY, USAAbstract: Radioimmunotherapy using a radiolabeled monoclonal antibody that targets tumor cells has been shown to be efficient for the treatment of many malignant cancers, with reduced side effects. However, the blood–brain barrier (BBB) inhibits the transport of intravenous antibodies to tumors in the brain. Recent studies have demonstrated that focused ultrasound (FUS) combined with microbubbles (MBs) is a promising method to transiently disrupt the BBB for the drug delivery to the central nervous system. To find the optimal FUS and MBs that can induce reversible increase in the BBB permeability, we employed minimally invasive multiphoton microscopy to quantify the BBB permeability to dextran-155 kDa with similar molecular weight to an antibody by applying different doses of FUS in the presence of MBs with an optimal size and concentration. The cerebral microcirculation was observed through a section of frontoparietal bone thinned with a micro-grinder. About 5 minutes after applying the FUS on the thinned skull in the presence of MBs for 1 minute, TRITC (tetramethylrhodamine isothiocyanate)-dextran-155 kDa in 1% bovine serum albumin in mammalian Ringer’s solution was injected into the cerebral circulation via the ipsilateral carotid artery by a syringe pump. Simultaneously, the temporal images were collected from the brain parenchyma ~100–200 µm below the pia mater. Permeability was determined from the rate of tissue solute accumulation around individual microvessels. After several trials, we found the optimal dose of FUS. At the optimal dose, permeability increased by ~14-fold after 5 minutes post-FUS, and permeability returned to the control level after 25 minutes. FUS without MBs or MBs injected without FUS did not change the permeability. Our method provides an accurate in vivo assessment for the transient BBB permeability change under the treatment of FUS. The optimal FUS dose found for the reversible BBB permeability increase without BBB disruption is reliable and can be applied to future clinical trials. Keywords: antibody delivery, multiphoton microscopy, in vivo cerebral microvessel permeability, rat brain

Quantum efficiencies (QEs) of innovative hybrid solar cells fabricated using micro/mesoporous zinc (hydr)oxide and its graphite-based composites sensitized by semiconductor quantum dots (SQDs) are reported. High absorption coefficient of CdSe SQDs and the wide band gap of zinc (hydr)oxide and its composites with graphite oxide (GO) are essential to achieve solar cells of higher QEs. Hybrid solar cells are fabricated from zinc (hydr)oxide and its composites (with 2 and 5 wt.% of graphite oxides, termed as, ZnGO-2 and ZnGO-5, respectively) while using potassium iodide or perovskite as an electrolyte. A two-photon fluorescence (TPF) imaging technique was used to determine the internal structure of the solar cell device. The photocurrent and current-voltage measurements were used to measure short-circuit current and open-circuit voltage to calculate the fill factor and QE of these solar cells. The highest QE (up to ∼ 10.62% ) is realized for a ZnGO-2-based solar cell using potassium iodide as its electrolyte and the CdSe quantum dot as its sensitizer.

Two-photon (2P) excitation of the second singlet (S2) state of a group of fluorescent agents with near infrared emission was used to extend the optical excitation and imaging regime of 2P microscope into “tissue optical window” (650–1100 nm). As the first step to achieve deeper optical imaging, Chlorophyll a and Indocyanine green are investigated and demonstrated as imaging agents using 2P S2 excitation at 800 nm for microscope imaging at 685 nm. The salient feature is to drive both the 2P excitation and emission wavelengths of the imaging agents to fall into the “tissue optical window.”

The band gap energies of micro/meso-porous zinc (hydr)oxide and its composites with 2 wt. % and 5 wt. % graphite oxides are reported using three optical characterization techniques. The obtained energy gaps (from 2.84 eV to 2.95 eV) of the composites are smaller than that for zinc oxide (∼3.2 eV) and zinc (hydr)oxide (∼3.06 eV). The band gap narrowing of the composite materials is due to the presence of defects, larger particle size, and weaker confinement. The bonds between zinc (hydr)oxide lattice and the carbon of graphene phase also contribute to this phenomenon. The structural properties of these materials are presented using Transmission Electron Microscopy, Scanning Tunneling Electron Microscopy, X-Ray analysis, and Two-Photon Fluorescence imaging Microscopy.

The optical properties of zinc (hydr)oxide and its porous composites with 2% and 5% graphite oxide (GO), thus forming ZnGO-2 and ZnGO-5, are investigated using reflectance spectroscopy and two-photon fluorescence (TPF) imaging. The bandgap energies for the Zn(OH)(2), ZnGO-2, and ZnGO-5 samples are determined to be in the range between 2.88 and 3.60 eV. The size of light-emitting regions (~from 4.5 to 45 μm) and pore size (~from 20 to 255 μm) are measured using the TPF imaging technique.