Search

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.

We detail a setup capable of simultaneous phase measurements by digital holographic microscopy and solution of the transport of intensity equation. Agreement between the retrieved phases is shown for a microlens array.

Understanding cellular iron homeostasis is critical to understanding cancer cell survival and proliferation, as this process includes balancing iron uptake with storage and utilization. Iron-bound transferrin (holo-Tf) will bind to the transferrin receptor (TfR) at the cell surface and undergo endocytosis where iron is released into a mildly acidified endosome. Inside the endosome the iron is reduced for transport across the membrane for utilization or into the cytosol for storage in ferritin (Ft). It remains unclear whether iron uptake and storage regulation remain coordinated processes in breast cancer cells. Normally, it is expected for TfR and Ft protein expression to be inversely related based on their regulation via iron regulatory proteins (IRP1/2); however, increased expression of both TfR and Ft have been expressed in heterogenous breast cancer populations. To address the heterogeneous populations, single-cell analysis with Raman hyperspectral imaging could evaluate the relationship of iron uptake and storage through identification of iron-bound Tf and Ft in unlabeled cells. Raman hyperspectral imaging at 532 nm excitation has facilitated the imaging of iron-bound Tf in unlabeled cells. It indicated disrupted Tf iron-release in triple-negative breast cancer cells (MDAMB231), but not in the luminal A breast cancer line (T47D). Our data suggests that 532 nm excitation of Ft results in unique spectra. Currently we are collecting data on the unlabeled breast cancer cells to determine the relationship of iron-bound Tf and Ft by single-cell Raman hyperspectral imaging. This method will accelerate our understanding of iron homeostasis in breast cancer cells.

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.

Iron is highly regulated in the body, since it is an essential element required for life. Fundamental understanding of the key processes that underlie the intracellular transport of iron will have a decisive impact on advancing treatment of diseases that are caused by iron deficiency and iron overload, e.g., anemias and hereditary hemochromatosis. Improved knowledge of iron intracellular transport will also provide insight into many other diseases where iron modulates the pathogenic process, e.g., metabolic syndrome, diabetes, neurodegenerative diseases, and cancer. Measuring the iron-bound form of transferrin in intact biological samples remains a technical challenge that needs to be overcome to understand regulation of endosomal iron release in cells and tissues. Serum transferrin (Tf) is a key regulator of systemic and cellular iron transport. Tf binds ferric iron (Fe3+) for transport throughout the body and delivery into cells via the transferrin receptor (TfR). The iron-bound Tf-TfR complex is endocytosed, and upon acidification of early endosome, the iron is released. Importantly, disruption of iron homeostasis has been linked to cancer progression. Although iron transport has been studied in detail, measurements of iron-bound Tf in tumor tissues are still lacking. Previously, we have developed and validated a Raman hyperspectral imaging technique that identified the iron-bound Tf peak at ~1300 cm-1 Raman shift. Here, we further investigate the variation in peak intensity within frozen tissue sections of T47D and MDA-MB-231 breast cancer tumor xenografts, which represent luminal and basal cancers, respectively. Our results indicate that Raman spectral imaging can be used to evaluate the iron-bound form of Tf in xenograft sections. Measurements of iron-bound Tf in tumor tissues will permit further characterization of iron transport in breast cancer.

Iron is an essential element required for human life, and is highly regulated in the body. Iron deficiency leads to many adverse health effects, such as anemias. The exact mechanisms of iron release in cells are not well known. We developed a Raman micro-spectroscopy technique that allows detection of transferrin (Tf) bound iron inside intact human cells. Ferric iron (Fe3+) bound to serum Tf is internalized into cells via the transferrin receptor (TfR). Methods that allow determining when and where Tf releases iron inside a cell lead to a better understanding of disease progression, including cancer. We have previously shown that Raman micro-spectroscopy is able to detect and quantify the Tf-bound iron in human breast cancer T47D cells. In this work, we applied hyperspectral Raman imaging to visualize the spatial distribution of Tf-bound iron in human breast cancer T47D and MDAMB231 cells internalized with iron-loaded Tf. We have also shown that Raman imaging can quantify the amount of iron under different times of Tf internalization prior to fixation. Raman microspectroscopy provides a unique way to determine the amount of iron under different Tf internalization times by employing the Raman metric, which was used to quantify iron content in iron bound Tf (holo-Tf) samples. Importantly, Raman microspectroscopy can be used to follow iron release from Tf in breast cancer cells. Determining the kinetics and location of iron release in cancer cells is key to further our understanding of iron metabolism during cancer progression.

Repair or reconstruction of organs is the goal of regenerative medicine. Bioengineered organoids that can differentiate when implanted in-vivo to partially restore organ function are being developed. Potentially, such organoids can be used to treat many medical conditions. A non-invasive method for quality monitoring of tissue engineered constructs is needed in order to ensure that they are ready for implantation. Raman micro-spectroscopy offers a way to quantitatively analyze cells and tissues without sample preparation or labelling dyes, which are not allowed in constructs used for the human implantation. Epithelial progenitor cells are parts of the complex organoids derived from the embryonic salivary gland cells. We have collected Raman spectra of the epithelial (acinar and ductal) cells treated with Fibroblast Growth Factor 2 (FGF2) and grown in organoids ex vivo over a period of (1 - 7 days). Evolution of the organoids over time was detected with Raman. These modifications, corresponding to the C-C stretch and C-H bend in proteins, as well as alterations in the Amide I and III envelopes, likely may correlate with changes in the cell environment or their differentiation state. Our goal is to develop Raman metrics that can be applied to the non-invasive monitoring of organoids.

Quantitative phase imaging (QPI) provides a label free method for imaging live cells and allows quantitative estimates of cell volume. Because the phase of light is not directly measurable at an imaging sensor, QPI techniques involve both hardware and software steps to reconstruct the phase. Digital holographic microscopy (DHM) is a QPI technique that utilizes an interferometer to combine a reference beam with a beam that passes through a specimen. This produces an interference pattern on the image sensor, and the specimen’s phase can be reconstructed using diffraction algorithms. One limitation of DHM is that the images are subject to coherent diffraction artifacts. Transport of intensity (TIE) method, on the other hand, uses the fact that defocused images of a specimen depend on the specimen’s phase to determine the phase from two or more defocused images. Its benefit over DHM is that it is compatible with conventional bright field imaging using sources of relatively low coherence. Although QPI methods can be compared on a variety of static phase targets, these largely consist of phase steps rather than the phase gradients present across cells. In order to compare the QPI methods described above on live cells, rapid switching between QPI modalities is required. 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, which allows the comparison of the accuracy of both measurements.

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.

Iron is an essential element required for human life. Iron is highly regulated in the body, as iron deficiency leads to many adverse health effects, such as anemias. Ferric iron (Fe3+) bound to serum transferrin (Tf) is internalized into cells via the transferrin receptor (TfR). Since the exact mechanisms of iron release in cells are not well known, a technique that allows detection of Tf bound iron inside intact human cells has been developed. Methods to determine when and where Tf releases iron inside a cell are required to better understand disease progression, including cancer. We have previously shown that Raman micro-spectroscopy is able to detect and quantify the Tf-bound iron in epithelial cells. In this work, we applied hyperspectral Raman imaging to visualize the spatial distribution of Tf-bound iron in human breast cancer T47D cells internalized with iron-loaded Tf, oxalate-Tf, a chemical Tf mutant unable to release iron, and iron-depleted Tf. We have also shown that Raman imaging can quantify the amount of iron under different times of Tf internalization (Tf uptake time), prior to fixation. Raman micro-spectroscopy provides a unique way to determine the amount of iron under different Tf internalization times by employing the Raman metric, which was used to quantify iron content in oxa- , apo-, and holo-Tf samples. Importantly, Raman microspectroscopy can be used to follow iron release from Tf in breast cancer cells. Determining the kinetics and location of iron release in cancer cells is key to further our understanding of iron metabolism during cancer progression.

Regenerative medicine encompasses the rebuilding or repairing of organs. We are developing bioengineered organoids that will differentiate when implanted in vivo to partially restore organ function. These complex organoids, derived from embryonic salivary gland cells, include both primary mesenchyme and epithelial progenitor cells. Noninvasive quality monitoring of tissue-engineered constructs is required before implantation of bioengineered constructs in vivo. Raman spectroscopy offers fast, simple, and, most importantly, non-invasive quantitative cell and tissue analysis that does not require elaborate sample preparation. We demonstrate the application of Raman micro-spectroscopy technique to in vitro monitoring of cell types within 3D cell clusters, with the ultimate goal of applying this technology in situ to monitor adult cell-derived organoids that are implanted in vivo. We have collected Raman spectra of epithelial and mesenchymal progenitor cells in vitro, and have shown that we are able to identify different Raman signatures corresponding to each cell type. In particular, we have observed Raman spectral differences which correspond to the C-C and C-N stretch in proteins, as well as in the Amide I and III envelopes. The embryonic mesenchyme cells are similar to mesenchymal stem cells, MSCs, which can differentiate into bone, cartilage, and other cell types. In addition to salivary gland tissue engineering applications, mesenchymal cells offer a great potential in repairing bone, cartilage, and damaged heart cells, and to treat inflammation and immune system diseases. In future studies, our Raman spectroscopy methods can be broadly applied to monitoring of organoids for application in many diseases.

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.

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.

Digital holographic microscopy uses interference patterns produced by the object and reference waves to computationally reconstruct both amplitude and phase of light reflected from a sample under study. The phase information recorded for each pixel can be converted to a height profile map, yielding a three-dimension image of the sample. Holographic imaging of layered structures, where layers are separated from one another by the axial distances exceeding the wavelength of imaging light, is challenging. Software based 2π phase discontinuity unwrapping, which relies on the gradients produced by the slowly varying sloped surfaces in the sample, is generally impossible. Additionally, dual wavelength phase unwrapping is complicated by the fact that if the layers are not sufficiently reflective, the unwrapping based on the comparison of two single wavelength phase images is unreliable. We present the design of a simultaneous dual wavelength digital holographic microscope, where the phase imaging of each individual layer is performed by a single wavelength, and then the axial distance between all layers is determined based on the comparison between the phase maps produced by each wavelength. By combining two interferometers within one setup, we could acquire two phase profiles simultaneously, enabling fast measurements. We demonstrate that this method is particularly well-suited for imaging of multilayered electrode structures embedded in glass, which contain both high and low reflectivity features.

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.

A fast, convenient way to determine the age of bones and ivories is important both in forensics and for classifying art objects in collections of art experts, restorers, art galleries and museums. Knowing the age of elephant tusks is also essential because there are many date-specific regulations of ivory trade. Radiocarbon dating is the standard method used to determine the age of organic materials, but it is expensive, time consuming, and damages the sample in the process. Raman spectroscopy is sensitive to rotational and vibrational molecular transitions, and also intermolecular vibrations. Therefore, it can provide information about sample make up, such as proteins and minerals, as well as detect spectral signatures associated with structural changes in molecules. Since Raman spectroscopy identifies the molecular bonds present in a sample, it is often used to determine its chemical composition. Bones and ivories contain two primary components: collagen and bioapatite. As the protein collagen deteriorates with time, its Raman signal decreases. The ratio of collagen-to-bioapatite peaks, therefore, is smaller in the older samples compared to the younger ones, providing a basis for sample dating. We employed Raman spectroscopy to non-destructively determine the age of several elephant tusk fragments. We have also used it to identify ivory imitations made of vegetable and plastic materials. Such materials have entirely different chemical composition, and their spectra are easily distinguished from those of bone and ivory. Peak fitting was employed to determine collagen and bioapatite components.

Sustained delivery of growth factors, proteins, drugs and other biologically active molecules is necessary for tissue engineering applications. Electrospun fibers are attractive tissue engineering scaffolds as they partially mimic the topography of the extracellular matrix (ECM). However, they do not provide continuous nourishment to the tissue. In search of a biomimetic scaffold for salivary gland tissue regeneration, we previously developed a blend nanofiber scaffold composed of the protein elastin and the synthetic polymer polylactic-co-glycolic acid (PLGA). The nanofiber scaffold promoted in vivo-like salivary epithelial cell tissue organization and apicobasal polarization. However, in order to enhance the salivary cell proliferation and biomimetic character of the scaffold, sustained growth factor delivery is needed. The composite nanofiber scaffold was optimized to act as a growth factor delivery system using epidermal growth factor (EGF) as a model protein. The nanofiber/EGF hybrid nanofibers were synthesized by double emulsion electrospinning where EGF is emulsified within a water/oil/water (w/o/w) double emulsion system. Successful incorporation of EGF was confirmed using Raman spectroscopy. EGF release profile was characterized using enzyme-linked immunosorbent assay (ELIZA) of the EGF content. Double emulsion electrospinning resulted in slower release of EGF. We demonstrated the potential of the proposed double emulsion electrospun nanofiber scaffold for the delivery of growth factors and/or drugs for tissue engineering and pharmaceutical applications.

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.

Non-invasive live cell measurements are an important tool in biomedical research. We present a combined digital holography/Raman spectroscopy technique to study live cell cultures during apoptosis. Digital holographic microscopy records an interference pattern between object and reference waves, so that the computationally reconstructed holographic image contains both amplitude and phase information about the sample. When the phase is mapped across the sample and converted into height information for each pixel, a three dimensional image is obtained. The measurement of live cell cultures by digital holographic microscopy yields information about cell shape and volume, changes to which are reflective of alterations in cell cycle and initiation of cell death mechanisms. Raman spectroscopy, on the other hand, is sensitive to rotational and vibrational molecular transitions, as well as intermolecular vibrations. Therefore, Raman spectroscopy provides complementary information about cells, such as protein, lipid and nucleic acid content, and, particularly, the spectral signatures associated with structural changes in molecules. The cell cultures are kept in the temperature-controlled environmental chamber during the experiment, which allows monitoring over multiple cell cycles. The DHM system combines a visible (red) laser source with conventional microscope base, and LabVIEW-run data processing. We analyzed and compared cell culture information obtained by these two methods.

Toxic and hazardous contaminants in household water present a significant health risk. We are developing a low cost, automated, multi-channel integrated system for in-line monitoring of common water contaminants, and are demonstrating preliminary results.

Understanding the mechanisms of cellular iron acquisition, storage, and excretion is critical for improving our knowledge of cell functioning. We applied Raman spectroscopic technique to non-invasively detect the presence of the iron content in protein mixture and cells.

Raman scattering microscopy is a powerful imaging technique used to identify chemical composition, structural and conformational state of molecules of complex samples in biology, biophysics, medicine and materials science. In this work, we have shown that Raman techniques allow the measurement of the iron content in protein mixtures and cells. Since the mechanisms of iron acquisition, storage, and excretion by cells are not completely understood, improved knowledge of iron metabolism can offer insight into many diseases in which iron plays a role in the pathogenic process, such as diabetes, neurodegenerative diseases, cancer, and metabolic syndrome. Understanding of the processes involved in cellular iron metabolism will improve our knowledge of cell functioning. It will also have a big impact on treatment of diseases caused by iron deficiency (anemias) and iron overload (hereditary hemochromatosis). Previously, Raman studies have shown substantial differences in spectra of transferrin with and without bound iron, thus proving that it is an appropriate technique to determine the levels of bound iron in the protein mixture. We have extended these studies to obtain hyperspectral images of transferrin in cells. By employing a Raman scanning microscope together with spectral detection by a highly sensitive back-illuminated cooled CCD camera, we were able to rapidly acquire and process images of fixed cells with chemical selectivity. We discuss and compare various methods of hyperspectral Raman image analysis and demonstrate the use of these methods to characterize cellular iron content without the need for dye labeling.

We present a three-dimensional microscopic technique based on digital holographic imaging, which allows highly accurate axial localization of features inside of a three dimensional sample. When a light wave is propagating through, or reflecting from, a microscopic object, the phase changes can be converted into intensity variations using the existing digital microscopic techniques. The phase change indicates the change in the optical path length, which can be then converted to physical thickness, providing the sample height information. This property of holograms is used in phasecontrast techniques, and can also be used for quantitative 3D imaging. However, if the sample contains features with different indices of refraction, this method can only provide the overall optical thickness, and cannot determine where in the axial direction the particular feature is located. As a result, the application of Digital Holographic Microscopy to imaging of organelles within live cells, or defects within semiconductor substrates, is limited to overall morphology of the sample. To determine the axial location of features inside of a three dimensional sample, we developed a phase image processing method based on analyzing images taken from non-zero incident angles. When compared, these images can discriminate between various axial depths of features, while still retaining the information about the overall thickness profile of the sample.

This is a study of the central nervous system (CNS) cells, including brain micro vascular endothelial cells (BMV) that constitute the blood brain barrier, and C6 glial cells that are the predominant cell in the brain. The cells are exposed to various chemicals by non-invasive, label-free methods. Digital holographic microscopy (DHM) is a technique that records an interference pattern between an object and reference waves, so that the computationally reconstructed holographic image contains both amplitude and phase information, and 3D images are obtained. The measurement of cell cultures by digital holographic microscopy yields information about cell death mechanisms, since these processes are correlated with individual cell volume. Our in-house DHM combines a visible (red) laser source with a conventional microscope base, and LabVIEW-run data processing. Terahertz spectral signatures are associated with structural changes in molecules and provide complementary information about cells. Both CNS cells BMV and C6 cells are treated with the drug “Methamphetamine" (METH), which induces apoptosis in neuronal cells and exhibits decrease in cell volume, a characteristic of cells undergoing apoptosis (induced cell death). METH can cause CNS cell death by cross-talk between mitochondria-, endoplasmic reticulum-, and receptor-mediated apoptotic events, all of which results in drug induced changes in neuroplasticity and significant neuropathology. Doxorubicin (DOX), a popular anticancer drug, is used as a control. We observe that METH treatment resulted in more pronounced cell volume shrinkage in both the BMV and C6 cells, as compared to DOX-induced cell apoptosis.

We present a three-dimensional microscopic technique based on simultaneous dual wavelength digital holography. In digital holographic microscopy, interference patterns produced by an object and reference waves are recorded by a camera. The computationally reconstructed holographic images contain the information about both amplitude and phase of the light reflected from the object. Phase is then mapped across the sample and converted into height information for each pixel. This technique was applied to imaging of electrodes embedded into glass substrates, which allowed three-dimensional reconstruction of their structure. Holographic imaging of the embedded layered structures, where each layer can be separated from the others by axial distances exceeding multiple wavelengths of imaging light, is difficult, because software phase unwrapping is practically impossible. The use of two wavelengths enables accurate axial measurements of multiple layers by comparing the phase maps produced by each individual wavelength. We demonstrated that the correct choice of wavelengths maximizes the axial range, at which an unambiguous 3D imaging can be performed. This provides not just three-dimensional structure of each layer, but also allows for height differentiation of layers. By employing wavelength cutoff filters, we were able to obtain the phase maps simultaneously, enabling fast measurements. We also developed a background removal technique, based on the quality of interference fringe pattern, which suppresses low intensity signal when no reliable phase information can be extracted. We showed that this is especially useful for multilayered embedded electrode structures, where each sample consists of both high and low reflectivity features.

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.

Methamphetamine induces neurotoxic responses in human brain that can further deteriorate into neural damage and cellular apoptosis. We examined the apoptosis inducing effects of Methamphetamine in real time in glial cells by digital holographic microscopy.

We measured the spontaneous-emission branching fractions of 46 levels in Nd II, selectively populated via single-frequency laser excitation of a 10 keV ion beam. The levels studied had term energies up to 29 955 cm–1, and decay branches with spontaneous emission in the range 372–850 nm were detected. The experimental accuracy for branching fractions over 0.1 was ~7%. We used these branching fractions along with our previously determined radiative lifetimes to infer transition probabilities and oscillator strengths for 430 transitions, which are useful for stellar abundance determinations. PACS Nos.: 32.70Cs, 95.30Ky

We have conducted studies of deep UV laser-induced fluorescence (LIF) for the reagentless detection of trace species and Dissolved Organic Compounds (DOC's) in water. Our LIF detection system had two interchangeable UV lasers, 266 nm and 355 nm, illuminating a flow cell containing a water sample. The fluorescence emitted at 90 degrees to the laser beam was collected by focusing optics, passed through cut-off and interference filters with 21 optical bandpass channels (240–680 nm ), and detected by a photomultiplier tube (PMT). The samples analyzed by the system included bottled, tap and river water; we have also worked with biological and chemical species (Bacillus Globigii, malathion). In terms of the excitation wavelength, it was observed that the deep UV excitation resulted in spectra that contained more features, and had better separation of the LIF from the Raman peak, thus enhancing the detection of unique spectral features.

Determination of optical properties (absorption (μa) and scattering (μs') coefficients) in human tissue is important when it comes to accurate calculation of fluence rate in and around tissue area. ALA application to the tissue induces production of protoporphyrin IX when activated by red light. Changes in the tissue optical properties can send information such as treatment outcome and tissue drug concentration. Patients in this study were treated with PDT for head and neck mucosal dysplasia. They were enrolled in a phase I study of escalating light doses and oral ALA with 60mg/kg. Red light at 630nm was administered to the tumor from a laser. The light dose was escalated from 50-200J/cm2 with a measured fluence rate at tissue surface of 100mW/cm2. We developed a light detection device for the purpose of determining optical properties in vivo using the semi-infinite method. The light detection device consists of two parallel, placed 5mm apart. In one of the catheters a 2 mm long linear diffusing light source is placed while in the second catheter, a calibrated isotropic detector is placed. The detector is scanned along the length of the light source containing catheter. Scans are done with the device placed on the treatment area (tumor) and on the normal tissue. Optical properties were measured in-vivo before and after PDT delivery for both normal tissue and tumor.

We measured the spontaneous-emission branching ratios of 69 levels in Sm II selectively populated via single-frequency laser excitation of a 10 keV ion beam. The levels studied had term energies up to 29 600 cm–1, and decay branches with spontaneous emission in the range 250–850 nm were detected. The experimental accuracy was in the range of 10%. We used these branching ratios along with our previously determined radiative lifetimes to infer transition probabilities and oscillator strengths for 608 transitions in the wavelength range 363–771 nm, which are useful for stellar abundance determinations.PACS Nos.: 32.70.Cs, 95.30.Ky

We have measured the isotope shifts of 87 transitions in Sm II in the 418–465 nm spectral region and the magnetic-dipole and electric-quadrupole hyperfine constants of 13 low-lying even-parity metastable levels and 76 odd-parity levels. These results will be immediately applicable to stellar spectroscopy.PACS Nos.: 32.30.–r, 32.10.–f

We have measured the radiative lifetimes of 33 levels of Pr II and 13 levels of Nd II using two variants of the beam-laser method. The levels studied had term energies up to ~30 000 cm–1 and lifetimes in the range 6–170 ns. Experimental accuracy was in the range 1–11%. We have used these lifetimes to update transition probabilities for 82 transitions in Pr II in the wavelength range 392–640 nm, which are useful for stellar abundance determinations. PACS Nos.: 32.70Cs, 95.30Ky

In the operating room, time is extremely precious, and the speed of one’s data acquisition system often determines whether the data will be taken or not. Our multichannel robotic platform addresses this issue by optimizing source and detector scanning procedures. Up to 16 fibers can be moved independently with resolution of 0.05 mm and speed of 50 mm/s using motors with position feedback. The initial fiber alignment employs a light beam/optical detector system for identical positioning of all motors. Peak and edge detection algorithms, for point and linear sources, are used with multiple fibers simultaneously for fast realignment of sources and detectors. The robotic platform is used to perform Diffuse Optical Tomography (DOT) measurements in solid prostate phantoms with both homogenous and inhomogeneous Optical Properties (OP). Correct positioning is critical for the accurate recovery of the OP. The light fluence rate distribution is determined by scanning multiple detector fibers simultaneously along lit linear sources placed throughout the phantom volume inside catheter needles. The scanning time for the entire DOT is about 10 seconds after the initial alignment. The OP distribution reconstruction is based on the steady-state light diffusion equation. The inverse interstitial DOT problem is solved using NIRFAST. The optical properties are recovered by iterative minimization of the difference between measured and calculated light fluence rates. Recovered OP agree with the actual values within 10%. The OP corrections are used to significantly improve light fluence accuracy for the entire volume of bulk tumor.

In-vivo light dosimetry for patients undergoing photodynamic therapy (PDT) is critical for predicting PDT outcome. Patients in this study are enrolled in a Phase I clinical trial of HPPH-mediated PDT for the treatment of non-small cell lung cancer with pleural effusion. They are administered 4mg per kg body weight HPPH 48 hours before the surgery and receive light therapy with a fluence of 15-45 J/cm2 at 661 and 665nm. Fluence rate (mW/cm2) and cumulative fluence (J/cm2) are monitored at 7 sites during the light treatment delivery using isotropic detectors. Light fluence (rate) delivered to patients is examined as a function of treatment time, volume and surface area. In a previous study, a correlation between the treatment time and the treatment volume and surface area was established. However, we did not include the direct light and the effect of the shape of the pleural surface on the scattered light. A real-time infrared (IR) navigation system was used to separate the contribution from the direct light. An improved expression that accurately calculates the total fluence at the cavity wall as a function of light source location, cavity geometry and optical properties is determined based on theoretical and phantom studies. The theoretical study includes an expression for light fluence rate in an elliptical geometry instead of the spheroid geometry used previously. The calculated light fluence is compared to the measured fluence in patients of different cavity geometries and optical properties. The result can be used as a clinical guideline for future pleural PDT treatment.

A custom-made robotic multichannel platform for interstitial photodynamic therapy (PDT) and diffuse optical tomography (DOT) was developed and tested in a phantom experiment. The system, which was compatible with the operating room (OR) environment, had 16 channels for independent positioning of light sources and/or isotropic detectors in separate catheters. Each channel’s motor had an optical encoder for position feedback, with resolution of 0.05 mm, and a maximum speed of 5 cm/s. Automatic calibration of detector positions was implemented using an optical diode beam that defined the starting position of each motor, and by means of feedback algorithms controlling individual channels. As a result, the accuracy of zero position of 0.1 mm for all channels was achieved. We have also employed scanning procedures where detectors automatically covered the appropriate range around source positions. Thus, total scan time for a typical optical properties (OP) measurement throughout the phantom was about 1.5 minutes with point sources. The OP were determined based on the measured light fluence rates. These enhancements allow a tremendous improvement of treatment quality for a bulk tumor compared to the systems employed in previous clinical trials.

Photosensitizer fluorescence emitted during photodynamic therapy (PDT) is of interest for monitoring the local concentration of the photosensitizer and its photobleaching. In this study, we use Monte Carlo (MC) simulations to evaluate the relationship between treatment light and fluorescence, both collected by an isotropic detector placed on the surface of the tissue. In treatment of the thoracic and peritoneal cavities, the light source position changes continually. The MC program is designed to simulate an infinitely broad photon beam incident on the tissue at various angles to determine the effect of angle. For each of the absorbed photons, a fixed number of fluorescence photons are generated and traced. The theoretical results from the MC simulation show that the angle theta has little effect on both the measured fluorescence and the ratio of fluorescence to diffuse reflectance. However, changes in the absorption and scattering coefficients, μa and μs′, do cause the fluorescence and ratio to change, indicating that a correction for optical properties will be needed for absolute fluorescence quantification. Experiments in tissue-simulating phantoms confirm that an empirical correction can accurately recover the sensitizer concentration over a physiologically relevant range of optical properties.

Current methods for the analysis of drinking water and many other liquids often call for the use of reagents and may require extensive sample preparation (American Public Health Association, 1989). For the case of water supplies and water treatment plants, this analysis is usually carried out once every few days or weeks (Killinger & Sivaprakasam, 2006). Most of the analysis is usually conducted using classical analytical chemical techniques, such as mass spectrometry, liquid chromatography, or fluorescence based or tagged reagents (Crompton, 2000). These analytical techniques are sensitive and provide accurate assessment of the chemistry related to the quality of the liquids. However, they often take considerable time and are usually not performed in real-time, especially for the case of a flowing process line. On the other hand, previous fluorescence spectroscopic measurements of ocean water by Coble showed that deep-UV excitation of naturally occurring organic compounds in water can yield significant and unique fluorescence signals in the near UV to visible wavelength range without the need to use additional reagents or sample preparation (Coble, 1996; Coble, 2007). As a result, we have been studying deep-UV laser-induced-fluorescence techniques for the detection of trace species in water and other liquids with the goal of using the natural fluorescence of trace species in the water or liquid samples and being able to provide readings within the time span of a few seconds. Toward this goal, we have developed a reagentless deep-UV laser and UV-LED induced fluorescence (LIF) system to detect and continuously observe in real time trace levels of colored dissolved organic matter (CDOM) or Dissolved Organic Compounds (DOCs) in water and distilled spirits, such as drinking water, and related water/alcohol based liquids with a sensitivity exceeding that of commercial spectrofluorometers. Our system has been used to detect ppb trace levels of plasicizer Bisphenol-A (BPA) that have leached into drinking water, and has detected and monitored trace levels of DOCs within ocean currents (Killinger & Sivaprakasam, 2006; Sivaprakasam et al. 2003; Sivaprakasam & Killinger, 2003). Recently, our LIF system has been used to measure fluorescence of reverse osmosis processed water and different types of drinking water (Sharikova & Killinger, 2007; Sharikova & Killinger, 2010). These LED/LIF applications have now been extended to additional water related samples, including humic acid samples, tannic acid and chlorinated water samples, juices, coffee, and several wines and distilled spirits; these recent results are presented in this paper.

We have developed a laser-induced fluorescence (LIF) system to detect and continuously observe in real time the levels of colored dissolved organic matter (CDOM) or Dissolved Organic Compounds (DOCs) in water from various sources, such as tap water and reverse osmosis processed water. At the same time, we have studied deep-UV light emitting diodes (LEDs) as alternative light sources for our system, which would make the apparatus cheaper and more compact. Our portable LIF system had two interchangeable microchip Nd:YAG lasers, operating at 266 nm and 355 nm, as UV sources, and fluorescence was measured over the range of 260-680 nm. The fluorescence was collected at 90o to the laser beam. We have also studied deep-UV LEDs emitting between 265 nm and 355 nm as alternative sources of fluorescence excitation. The average laser power was approximately 30 times that of the LED. Fluorescence spectra from sea water, tap water, and reverse osmosis water for both excitation sources were also measured, and similar spectra were observed. Differences in the signal intensity due to the difference in the laser and LED excitation intensity were consistent with theory.

We have developed a deep-UV laser-induced fluorescence system for fluorescence detection of water-dissolved organic species. Deep-UV LEDs also were used as the excitation source. These systems have been employed for real-time monitoring of drinking water.

We have used a deep-UV laser-induced fluorescence system to monitor in real time organic species present in tap water. We have also tested a 265 nm UV-LED as an excitation source for our system.

A UV (266 nm) laser-induced fluorescence (LIF) system with high sensitivity has been used to record fluorescent spectra (300 nm - 700 nm) of various water samples, such as distilled, tap and river water. Large fluorescence peaks corresponding to the fluorescence of Dissolved Organic Compounds (DOCs) were observed in river samples. Significant differences in spectra between different brands of drinking and distilled bottled water were also observed. The LIF system is currently used to measure the trace species in water processed by Reverse Osmosis Water Purification Unit (ROWPU). Initial spectra of the input and output water are presented.

The monitoring of water contamination and trace species is of great importance. Our lab has developed two highly sensitive LIF detection systems currently used to detect traces of bacteria endospores and dissolved organic compounds in water.

The description of experimental installation for investigation fractal structure of dental tissues light scattering is presented. The installation includes light source (He-Ne laser), beam transformer based on microlens array, light polarization control unit and registrating device (which represented computer interfaced CCD-camera). The experimental installation provides the estimation of different kinds of light scattering in the enamel and dentin. The joint computer processing of images corresponding to different states light polarization allows us to separate the effects of light scattering caused by different scattering object as well as by relief junctions. The results of research may be useful for dental restorations, because fractal dimension defined adhesion properties of dentin.

Purpose: To calculate scatter factor (SF) for flattening filter free (FFF) fields using pencil‐beam convolution and equivalent square methods, and determine the accuracy of the predicted dose per MU in both cases by comparison with experimental data. Methods: Scatter factor and dose per MU were calculated for 6xFFF and 10xFFF linac Varian TrueBeam at the benchmark points using two methods. The equivalent square method assumed constant SF based on the field size. In the convolution method, the off‐axis scatter factor values were found by calculating a convolution of an extrapolated scatter kernel, based on measured data, with primary off‐axis ratio (POAR) values. Essentially, the equivalent square method assumed a flat beam profile, while the convolution method took into account the actual shape of the beam. Results: The normalized scatter factor values were up to 25% different between two methods. Since the convolution method took into account that FFF fields are non‐uniform, it was superior for the accurate FFF beam dose calculation. Conclusion: This work indicates the importance of precise scatter factor determination to accurately predict D/MU for FFF beams due to non‐uniform beam profile. The implication for secondary MU calculation will be discussed.