Raman spectroscopy based molecular signatures of methamphetamine and HIV induced mitochondrial dysfunction.

METH and HIV Tat treatment results in increased oxidative stress which affects cellular metabolism and causes DNA damage in the treated microglia. Both, METH ± HIV Tat impair mitochondrial respiration, leading to dysfunction in bioenergetics and increased ROS in microglial cells. Our data indicate that mitochondrial dysfunction may be key to the METH and/or HIV Tat-induced neuropathology. METH and/or HIV Tat induced changes in the protein, lipid and nucleotide concentration in microglial cells were measured by Raman Spectroscopy, and we speculate that these fundamental molecular-cellular changes in microglial cells contribute to the neuropathology that is associated with METH abuse in HIV patients.

Raman microspectroscopy fingerprinting of organoid differentiation state.

BACKGROUND: Organoids, which are organs grown in a dish from stem or progenitor cells, model the structure and function of organs and can be used to define molecular events during organ formation, model human disease, assess drug responses, and perform grafting in vivo for regenerative medicine approaches. For therapeutic applications, there is a need for nondestructive methods to identify the differentiation state of unlabeled organoids in response to treatment with growth factors or pharmacologicals. METHODS: Using complex 3D submandibular salivary gland organoids developed from embryonic progenitor cells, which respond to EGF by proliferating and FGF2 by undergoing branching morphogenesis and proacinar differentiation, we developed Raman confocal microspectroscopy methods to define Raman signatures for each of these organoid states using both fixed and live organoids. RESULTS: Three separate quantitative comparisons, Raman spectral features, multivariate analysis, and machine learning, classified distinct organoid differentiation signatures and revealed that the Raman spectral signatures were predictive of organoid phenotype. CONCLUSIONS: As the organoids were unlabeled, intact, and hydrated at the time of imaging, Raman spectral fingerprints can be used to noninvasively distinguish between different organoid phenotypes for future applications in disease modeling, drug screening, and regenerative medicine.

Mitochondrial Dynamics in SARS-COV2 Spike Protein Treated Human Microglia: Implications for Neuro-COVID.

Emerging clinical data from the current COVID-19 pandemic suggests that ~ 40% of COVID-19 patients develop neurological symptoms attributed to viral encephalitis while in COVID long haulers chronic neuro-inflammation and neuronal damage result in a syndrome described as Neuro-COVID. We hypothesize that SAR-COV2 induces mitochondrial dysfunction and activation of the mitochondrial-dependent intrinsic apoptotic pathway, resulting in microglial and neuronal apoptosis. The goal of our study was to determine the effect of SARS-COV2 on mitochondrial biogenesis and to monitor cell apoptosis in human microglia non-invasively in real time using Raman spectroscopy, providing a unique spatio-temporal information on mitochondrial function in live cells. We treated human microglia with SARS-COV2 spike protein and examined the levels of cytokines and reactive oxygen species (ROS) production, determined the effect of SARS-COV2 on mitochondrial biogenesis and examined the changes in molecular composition of phospholipids. Our results show that SARS- COV2 spike protein increases the levels of pro-inflammatory cytokines and ROS production, increases apoptosis and increases the oxygen consumption rate (OCR) in microglial cells. Increases in OCR are indicative of increased ROS production and oxidative stress suggesting that SARS-COV2 induced cell death. Raman spectroscopy yielded significant differences in phospholipids such as Phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE) and phosphatidylcholine (PC), which account for ~ 80% of mitochondrial membrane lipids between SARS-COV2 treated and untreated microglial cells. These data provide important mechanistic insights into SARS-COV2 induced mitochondrial dysfunction which underlies neuropathology associated with Neuro-COVID.

Automated quantification of vomeronasal glomeruli number, size, and color composition after immunofluorescent staining.

Glomeruli are neuropil-rich regions of the main or accessory olfactory bulbs (AOB) where the axons of olfactory or vomeronasal neurons and dendrites of mitral/tufted cells form synaptic connections. In the main olfactory system, olfactory sensory neurons (OSNs) expressing the same receptor innervate 1 or 2 glomeruli. However, in the accessory olfactory system, vomeronasal sensory neurons (VSNs) expressing the same receptor can innervate up to 30 different glomeruli in the AOB. Genetic mutation disrupting genes with a role in defining the identity/diversity of olfactory and vomeronasal neurons can alter the number and size of glomeruli. Interestingly, 2 cell surface molecules, Kirrel2 and Kirrel3, have been indicated as playing a critical role in the organization of axons into glomeruli in the AOB. Being able to quantify differences in glomeruli features, such as number, size, or immunoreactivity for specific markers, is an important experimental approach to validate the role of specific genes in controlling neuronal connectivity and circuit formation in either control or mutant animals. Since the manual recognition and quantification of glomeruli on digital images is a challenging and time-consuming task, we generated a program in Python able to identify glomeruli in digital images and quantify their properties, such as size, number, and pixel intensity. Validation of our program indicates that our script is a fast and suitable tool for high-throughput quantification of glomerular features of mouse lines with different genetic makeup.

Telomere Length Shortening in Microglia: Implication for Accelerated Senescence and Neurocognitive Deficits in HIV.

The widespread use of combination antiretroviral therapy (cART) has led to the accelerated aging of the HIV-infected population, and these patients continue to have a range of mild to moderate HIV-associated neurocognitive disorders (HAND). Infection results in altered mitochondrial function. The HIV-1 viral protein Tat significantly alters mtDNA content and enhances oxidative stress in immune cells. Microglia are the immune cells of the central nervous system (CNS) that exhibit a significant mitotic potential and are thus susceptible to telomere shortening. HIV disrupts the normal interplay between microglia and neurons, thereby inducing neurodegeneration. HIV cART contributes to the inhibition of telomerase activity and premature telomere shortening in activated peripheral blood mononuclear cells (PBMC). However, limited information is available on the effect of cART on telomere length (TL) in microglia. Although it is well established that telomere shortening induces cell senescence and contributes to the development of age-related neuro-pathologies, the effect of HIV-Tat on telomere length in human microglial cells and its potential contribution to HAND are not well understood. It is speculated that in HAND intrinsic molecular mechanisms that control energy production underlie microglia-mediated neuronal injury. TL, telomerase and mtDNA expression were quantified in microglial cells using real time PCR. Cellular energetics were measured using the Seahorse assay. The changes in mitochondrial function were examined by Raman Spectroscopy. We have also examined TL in the PBMC obtained from HIV-1 infected rapid progressors (RP) on cART and those who were cART naïve, and observed a significant decrease in telomere length in RP on cART as compared to RP's who were cART naïve. We observed a significant decrease in telomerase activity, telomere length and mitochondrial function, and an increase in oxidative stress in human microglial cells treated with HIV Tat. Neurocognitive impairment in HIV disease may in part be due to accelerated neuro-pathogenesis in microglial cells, which is attributable to increased oxidative stress and mitochondrial dysfunction.

Iron-binding cellular profile of transferrin using label-free Raman hyperspectral imaging and singular value decomposition (SVD).

Serum transferrin (Tf) is the essential iron transport protein in the body. Transferrin is responsible for the sequestration of free iron in serum and the delivery of iron throughout the body and into cells, where iron is released inside a mildly acidified endosome. Altered iron distributions are associated with diseases such as iron-overload, cancer, and cardiovascular disease. The presence of free iron is linked to deleterious redox reactions, inside and outside cells and organelles. As Tf iron release is pH dependent, any changes in intraorganelle and extracellular pH, often associated with disease progression, could inhibit normal iron delivery or accelerate iron release in the wrong compartment. However, imaging approaches to monitor changes in the iron-bound state of Tf are lacking. Recently, Raman spectroscopy has been shown to measure iron-bound forms of Tf in solution, intact cells and tissue samples. Here, a biochemical Raman assay has been developed to identify iron-release from Tf following modification of chemical environment. Quantitative singular value decomposition (SVD) method has been applied to discriminate between iron-bound Tf samples during endocytic trafficking in intact cancer cells subjected to Raman hyperspectral confocal imaging. We demonstrate the strength of the SVD method to monitor pH-induced Tf iron-release using Raman hyperspectral imaging, providing the redox biology field with a novel tool that facilitates subcellular investigation of the iron-binding profile of transferrin in various disease models.

Circadian Modulation of Neurons and Astrocytes Controls Synaptic Plasticity in Hippocampal Area CA1.

Most animal species operate according to a 24-h period set by the suprachiasmatic nucleus (SCN) of the hypothalamus. The rhythmic activity of the SCN modulates hippocampal-dependent memory, but the molecular and cellular mechanisms that account for this effect remain largely unknown. Here, we identify cell-type-specific structural and functional changes that occur with circadian rhythmicity in neurons and astrocytes in hippocampal area CA1. Pyramidal neurons change the surface expression of NMDA receptors. Astrocytes change their proximity to synapses. Together, these phenomena alter glutamate clearance, receptor activation, and integration of temporally clustered excitatory synaptic inputs, ultimately shaping hippocampal-dependent learning in vivo. We identify corticosterone as a key contributor to changes in synaptic strength. These findings highlight important mechanisms through which neurons and astrocytes modify the molecular composition and structure of the synaptic environment, contribute to the local storage of information in the hippocampus, and alter the temporal dynamics of cognitive processing.

Quantitative label-free imaging of iron-bound transferrin in breast cancer cells and tumors.

Transferrin (Tf) is an essential serum protein which delivers iron throughout the body via transferrin-receptor (TfR)-mediated uptake and iron release in early endosomes. Currently, there is no robust method to assay the population of iron-bound Tf in intact cells and tissues. Raman hyperspectral imaging detected spectral peaks that correlated with iron-bound Tf in intact cells and tumor xenografts sections (~1270-1300 cm-1). Iron-bound (holo) and iron-free (apo) human Tf forms were endocytosed by MDAMB231 and T47D human breast cancer cells. The Raman iron-bound Tf peak was identified in cells treated with holo-Tf, but not in cells incubated with apo-Tf. A reduction in the Raman peak intensity between 5 and 30 min of Tf internalization was observed in T47D, but not in MDAMB231, suggesting that T47D can release iron from Tf more efficiently than MDAMB231. MDAMB231 may display a disrupted iron homeostasis due to iron release delays caused by alterations in the pH or ionic milieu of the early endosomes. In summary, we have demonstrated that Raman hyperspectral imaging can be used to identify iron-bound Tf in cell cultures and tumor xenografts and detect iron release behavior of Tf in breast cancer cells.

Dual wavelength digital holographic imaging of layered structures.

We present a dual wavelength digital holographic technique for three-dimensional microscopic imaging of layered structures, where layers are separated from one another by the axial distances exceeding the wavelength of imaging light. Our methodology not only provides the three-dimensional structure of each layer, but also allows the height differentiation of distinct layers. We have also implemented a technique suppressing low intensity signal when no reliable phase information can be extracted, based on the quality of the interference fringe pattern. We utilize a dual wavelength setup, where the combination of two overlapping interferometers enables simultaneous acquisition of two phase profiles. We demonstrate that this imaging modality is particularly well-suited for imaging of multilayered electrode structures embedded in glass.

Raman Spectroscopy Allows for the Determination of Elephant Ivory Age.

Determination of the age of ivory is important for controlling illegal trafficking and the proper identification of ivory artifacts. Radiocarbon dating is the standard method of determining the age of ivories; however, it requires the destruction of a fragment of the sample. Raman spectroscopy is a nondestructive technique, and therefore can be used on artwork. Moreover, Raman measurements can be done using a portable system, and the data analysis can be performed on the spot once the groundwork is done. Ivories contain two primary components: collagen and bioapatite. Raman spectrum of ivory material is mainly a sum of the vibrational bands of these components. As collagen deteriorates with time, its Raman signal decreases; therefore, the ratio of collagen to bioapatite peaks is smaller in the older samples compared to the younger ones, providing a basis for sample dating. We have compared the results of Raman and radiocarbon measurements applied to a set of elephant ivory fragments and have successfully calibrated the Raman data set using radiocarbon measurements. We found that the Raman collagen to bioapatite peak ratios of the samples can be used as a metric to determine their age, providing a nondestructive technique to assess the age of ivory samples. We have also used singular value decomposition (SVD) to analyze the whole Raman spectra. We have observed clear separation between samples of different ages in the SVD component space. The samples also tended to align along the timeline diagonal in the correct order. The changes in multiple collagen and bioapatite peaks contribute to the differences in Raman spectra of ivory samples of different age.

Comparative phase imaging of live cells by digital holographic microscopy and transport of intensity equation methods.

We describe a microscopic setup implementing phase imaging by digital holographic microscopy (DHM) and transport of intensity equation (TIE) methods, which allows the results of both measurements to be quantitatively compared for either live cell or static samples. Digital holographic microscopy is a well-established method that provides robust phase reconstructions, but requires a sophisticated interferometric imaging system. TIE, on the other hand, is directly compatible with bright-field microscopy, but is more susceptible to noise artifacts. We present results comparing DHM and TIE on a custom-built microscope system that allows both techniques to be used on the same cells in rapid succession, thus permitting the comparison of the accuracy of both methods.

Characterization of nanofibers for tissue engineering: Chemical mapping by Confocal Raman microscopy.

Nanofiber scaffolds are used in bioengineering for functional support of growing tissues. To fine tune nanofiber properties for specific applications, it is often necessary to characterize the spatial distribution of their chemical content. Raman spectroscopy is a common tool used to characterize chemical composition of various materials, including nanofibers. In combination with a confocal microscope, it allows simultaneous mapping of both spectral and spatial features of inhomogeneous structures, also known as hyperspectral imaging. However, such mapping is usually performed on microscopic scale, due to the resolution of the scanning system being diffraction limited (about 0.2-0.5 micron, depending on the excitation wavelength). We present an application of confocal Raman microscopy to hyperspectral mapping of nanofibers, where nanoscale features are resolved by means of oversampling and extensive data processing, including Singular Value Decomposition and Classical Least Squares decomposition techniques. Oversampling and data processing facilitated evaluation of the spatial distribution of different chemical components within multi-component nanofibers.

Methamphetamine-induced apoptosis in glial cells examined under marker-free imaging modalities.

We used phase microscopy and Raman spectroscopic measurements to assess the response of in vitro rat C6 glial cells following methamphetamine treatment in real time. Digital holographic microscopy (DHM) and three-dimensional (3-D) tomographic nanoscopy allow measurements of live cell cultures, which yield information about cell volume changes. Tomographic phase imaging provides 3-D information about the refractive index distribution associated with the morphology of biological samples. DHM provides similar information, but for a larger population of cells. Morphological changes in cells are associated with alterations in cell cycle and initiation of cell death mechanisms. Raman spectroscopy measurements provide information about chemical changes within the cells. Our Raman data indicate that the chemical changes in proteins preceded morphological changes, which were seen with DHM. Our study also emphasizes that tomographic phase imaging, DHM, and Raman spectroscopy are imaging tools that can be utilized for noninvasive simultaneous monitoring of morphological and chemical changes in cells during apoptosis and can also be used to monitor other dynamic cell processes.

Methamphetamine Induces Apoptosis of Microglia via the Intrinsic Mitochondrial-Dependent Pathway.

Methamphetamine (METH) is a drug of abuse, the acute and chronic use of which induces neurotoxic responses in the human brain, ultimately leading to neurocognitive disorders. Our goals were to understand the impact of METH on microglial mitochondrial respiration and to determine whether METH induces the activation of the mitochondrial-dependent intrinsic apoptosis pathway in microglia. We assessed the expression of pro- apoptosis genes using qPCR of RNA extracted from a human microglial cell line (HTHU). We examined the apoptosis-inducing effects of METH on microglial cells using digital holographic microscopy (DHM) to quantify real-time apoptotic volume decrease (AVD) in microglia in a noninvasive manner. METH treatment significantly increased AVD, activated Caspase 3/7, increased the gene expression levels of the pro- apoptosis proteins, APAF-1 and BAX, and decreased mitochondrial DNA content. Using immunofluorescence analysis, we found that METH increased the expression of the mitochondrial proteins cytochrome c and MCL-1, supporting the activation of mitochondrion-dependent (intrinsic) apoptosis pathway. Cellular bio-energetic flux analysis by Agilent Seahorse XF Analyzer revealed that METH treatment increased both oxidative and glycolytic respiration after 3 h, which was sustained for at least 24 h. Several events, such as oxidative stress, neuro-inflammatory responses, and mitochondrial dysfunction, may converge to mediate METH-induced apoptosis of microglia that may contribute to neurotoxicity of the CNS. Our study has important implications for therapeutic strategies aimed at preserving mitochondrial function in METH abusing patients.

Mesenchymal Cells Affect Salivary Epithelial Cell Morphology on PGS/PLGA Core/Shell Nanofibers.

Engineering salivary glands is of interest due to the damaging effects of radiation therapy and the autoimmune disease Sjögren's syndrome on salivary gland function. One of the current problems in tissue engineering is that in vitro studies often fail to predict in vivo regeneration due to failure of cells to interact with scaffolds and of the single cell types that are typically used for these studies. Although poly (lactic co glycolic acid) (PLGA) nanofiber scaffolds have been used for in vitro growth of epithelial cells, PLGA has low compliance and cells do not penetrate the scaffolds. Using a core-shell electrospinning technique, we incorporated poly (glycerol sebacate) (PGS) into PLGA scaffolds to increase the compliance and decrease hydrophobicity. PGS/PLGA scaffolds promoted epithelial cell penetration into the scaffold and apical localization of tight junction proteins, which is necessary for epithelial cell function. Additionally, co-culture of the salivary epithelial cells with NIH3T3 mesenchymal cells on PGS/PLGA scaffolds facilitated epithelial tissue reorganization and apical localization of tight junction proteins significantly more than in the absence of the mesenchyme. These data demonstrate the applicability of PGS/PLGA nanofibers for epithelial cell self-organization and facilitation of co-culture cell interactions that promote tissue self-organization in vitro.

Core/shell nanofiber characterization by Raman scanning microscopy.

Core/shell nanofibers are becoming increasingly popular for applications in tissue engineering. Nanofibers alone provide surface topography and increased surface area that promote cellular attachment; however, core/shell nanofibers provide the versatility of incorporating two materials with different properties into one. Such synthetic materials can provide the mechanical and degradation properties required to make a construct that mimics in vivo tissue. Many variations of these fibers can be produced. The challenge lies in the ability to characterize and quantify these nanofibers post fabrication. We developed a non-invasive method for the composition characterization and quantification at the nanoscale level of fibers using Confocal Raman microscopy. The biodegradable/biocompatible nanofibers, Poly (glycerol-sebacate)/Poly (lactic-co-glycolic) (PGS/PLGA), were characterized as a part of a fiber scaffold to quickly and efficiently analyze the quality of the substrate used for tissue engineering.

Quantification of Confocal Images Using LabVIEW for Tissue Engineering Applications.

Quantifying confocal images to enable location of specific proteins of interest in three-dimensional (3D) is important for many tissue engineering (TE) applications. Quantification of protein localization is essential for evaluation of specific scaffold constructs for cell growth and differentiation for application in TE and tissue regeneration strategies. Although obtaining information regarding protein expression levels is important, the location of proteins within cells grown on scaffolds is often the key to evaluating scaffold efficacy. Functional epithelial cell monolayers must be organized with apicobasal polarity with proteins specifically localized to the apical or basolateral regions of cells in many organs. In this work, a customized program was developed using the LabVIEW platform to quantify protein positions in Z-stacks of confocal images of epithelial cell monolayers. The program's functionality is demonstrated through salivary gland TE, since functional salivary epithelial cells must correctly orient many proteins on the apical and basolateral membranes. Bio-LabVIEW Image Matrix Evaluation (Bio-LIME) takes 3D information collected from confocal Z-stack images and processes the fluorescence at each pixel to determine cell heights, nuclei heights, nuclei widths, protein localization, and cell count. As a demonstration of its utility, Bio-LIME was used to quantify the 3D location of the Zonula occludens-1 protein contained within tight junctions and its change in 3D position in response to chemical modification of the scaffold with laminin. Additionally, Bio-LIME was used to demonstrate that there is no advantage of sub-100 nm poly lactic-co-glycolic acid nanofibers over 250 nm fibers for epithelial apicobasal polarization. Bio-LIME will be broadly applicable for quantification of proteins in 3D that are grown in many different contexts.

Live-cell quantification and comparison of mammalian oocyte cytosolic lipid content between species, during development, and in relation to body composition using nonlinear vibrational microscopy.

Cytosolic lipids participate in the growth, development, and overall health of mammalian oocytes including many roles in cellular homeostasis. Significant emphasis has been placed on the study of lipids as a dynamic organelle, which in turn requires the development of tools and techniques to quantitate and compare how lipid content relates to cellular structure, function, and normalcy. Objectives of this study were to determine if nonlinear vibrational microscopy (e.g., coherent anti-Stokes Raman scattering or CARS microscopy) could be used for live-cell imaging to quantify and compare lipid content in mammalian oocytes during development and in relation to body composition; and compare its efficacy to methods involving cellular fixation and staining protocols. Results of this study demonstrate that CARS is able to identify lipids in live mammalian oocytes, and there exists quantifiable and consistent differences in percent lipid composition across ooctyes of different species, developmental stages, and in relation to body composition. Such a method of live-cell lipid quantification has (i) experimental power in basic cell biology, (ii) practical utility for identifying developmental predictive biomarkers while advancing biology-based oocyte/embryo selection, and (iii) ability to yield rationally supporting technology for decision-making in rodents, domestic species, and human assisted reproduction and/or fertility preservation.

Human oral mucosa tissue-engineered constructs monitored by Raman fiber-optic probe.

In maxillofacial and oral surgery, there is a need for the development of tissue-engineered constructs. They are used for reconstructions due to trauma, dental implants, congenital defects, or oral cancer. A noninvasive monitoring of the fabrication of tissue-engineered constructs at the production and implantation stages done in real time is extremely important for predicting the success of tissue-engineered grafts. We demonstrated a Raman spectroscopic probe system, its design and application, for real-time ex vivo produced oral mucosa equivalent (EVPOME) constructs noninvasive monitoring. We performed in vivo studies to find Raman spectroscopic indicators for postimplanted EVPOME failure and determined that Raman spectra of EVPOMEs preexposed to thermal stress during manufacturing procedures displayed correlation of the band height ratio of CH2 deformation to phenylalanine ring breathing modes, giving a Raman metric to distinguish between healthy and compromised postimplanted constructs. This study is the step toward our ultimate goal to develop a stand-alone system, to be used in a clinical setting, where the data collection and analysis are conducted on the basis of these spectroscopic indicators with minimal user intervention.