It is a well-established fact that iron metabolism is disrupted in breast cancer cells. Assessment of iron transport and metabolism is necessary to understand molecular mechanism of breast cancer progression. Previously, Raman spectroscopy has been used to measure the Raman spectral profile of iron-bound proteins in breast cancer cells. By harnessing the principle of inelastic scattering of light, Raman spectroscopy offers a powerful, label-free, and nondestructive tool for determination of molecular structures and analysis of chemical bonds. The current study employed a specific experimental approach to capture shifts in the Raman signature of iron-binding proteins, such as transferrin. Focusing on cytoplasmic regions (exclusive of the nucleus) permits improved analysis of iron-binding proteins localized to vesicles present in the cytoplasm. The acquired spectra were subjected to rigorous analysis using singular value decomposition (SVD), a powerful mathematical technique that possesses the ability to reveal underlying trends and enhance biological analysis and interpretation. It involves detecting overlapping frequency patterns in the dataset. By applying SVD to distinguish the Raman spectral profiles of iron-bound transferrin in breast cancer cells, we obtained accurate results that have played a pivotal role in discerning and characterizing the Raman spectral profile of iron-bound transferrin in breast cancer cells.
Alginate is a natural polysaccharide found in brown algae and has a unique feature, the ability to form a hydrogel upon encountering Ca2+. Its exceptional characteristics make alginate hydrogels highly desirable for a range of biomedical applications, such as drug delivery, wound healing, and in particular, tissue engineering and cell therapy, where it is used as scaffolding or as a cell delivery vehicle. After using alginate hydrogel for cell delivery in vivo, one of our objectives was to specifically detect alginate in mouse tissue cryosections containing cell-scaffold constructs to evaluate scaffold cell-scaffold integration with host tissue and degradation. Due to difficulties encountered in detecting alginate using immunohistochemistry with mouse-derived antibodies, we aimed to develop an alternative method to definitively identify alginate within tissue cryosection samples using Raman spectroscopy. The Raman spectra of pure tissue had specific peaks convenient for identification. We identified a region where alginate consistently had stronger signal than either tissue or tissue freezing media. We also detected alginate-specific Raman peaks at 816, 888, 959, 1309, 1433 cm-1. By collecting the Raman spectra of the samples containing all three substances (alginate, freezing media, and tissue), analyzing them either by characteristic spectral peaks or classical least squares (CLS) method, and mapping the media, alginate, and tissue on the brightfield sample image, we were able to discriminate the alginate from tissue and freezing media. The notable sensitivity and specificity of Raman spectroscopy renders it a promising method for the identification of alginate and alginate-based materials in tissue engineering.
Programmed cell death, or apoptosis, can be triggered in C6 glial cells through exposure to the drug methamphetamine. Non-invasive, quantitative tracking of apoptotic glial cell morphology can be difficult, as many cellular samples are weakly scattering, and therefore traditional bright field images may be of low contrast. Higher contrast images may be found through incorporation of the quantitative phase delay a beam can undergo due to transmission through a sample. In addition, quantitative phase information can be used, non-invasively, to track meaningful morphological quantities over time. Digital holographic microscopy (DHM) and utilization of the transport of intensity equation (TIE) are two label-free, high-resolution phase imaging techniques. DHM quantitatively retrieves phase through measurement of a hologram, or the interference pattern created when combining object and reference beams. The TIE quantifies the relationship between a field’s phase and intensity upon propagation. Solving the TIE requires measurement of an in-focus intensity, and images in symmetric planes about focus. On a setup capable of simultaneous data collection for both techniques, phase reconstructions were retrieved of C6 rat glial cells undergoing methamphetamine induced apoptosis. The two techniques’ measurements of total optical volume of cell clusters were compared over time. Additionally, the behavior of cells’ index of refraction during apoptosis was explored through optical diffraction tomography (ODT) retrieved reconstructions. Through these reconstructions, both cell volume and cell optical volume were tracked. The average relative refractive index behavior measured by ODT was extended to extrapolate volume from the TIE/DHM optical volume measurements.
Cytochrome c, an essential protein integral to the electron transport chain within cellular mitochondria, plays a crucial role in the intricate process of apoptosis, or programmed cell death. An early event in apoptosis involves the release of cytochrome c from the mitochondria's intermembrane space into the cytoplasm. This paper explores the detection of cytochrome c during apoptosis using Raman spectroscopy, with a specific focus on its release from the mitochondria of human microglial cells (HTHμ). Raman spectroscopy, a non-invasive and label-free analytical technique, allows the examination of biomolecular changes based on their chemical properties. Our experimental approach induced apoptosis in HTHμ cells using methamphetamine (METH) and utilized Raman spectroscopy on both control and apoptotic samples. Through the analysis of spectra by singular value decomposition (SVD), which reveals subtle trends and facilitates biological interpretation, distinct spectral features corresponding to cytochrome c were identified. This evidence supports the concept of cytochrome c release from the mitochondria during apoptosis. The label-free nature and high sensitivity of Raman spectroscopy position it as a promising technique for studying apoptosis in biomedical research and contributing to the development of innovative diagnostic approaches for apoptotic-related disorders.
Raman hyperspectral imaging enables visualization and measurement of the distribution of iron-binding proteins, as well as assessment of the role of proteins involved in the regulation of intracellular iron transport and membrane trafficking in intact cells. To advance the mechanistic understanding of intracellular iron transport, we have performed Raman imaging in MDA-MB-231 wild-type breast cancer cells and compared them to CRISPR/Cas9-based knock-out cells of two intracellular iron homeostasis regulators: the Divalent Metal Transporter 1 (DMT1) and the small GTPase Rab4A. Multivariate singular value decomposition (SVD) analysis showed that the wild type vs. knockout cell populations of both genes could be separated into two distinct groups. Both DMT1 and Rab4A silencing have significant and distinct impacts in a variety of Raman spectra peaks, indicating a strong impact on cell metabolism. Label-free and non-invasive Raman imaging of DMT1 or Rab4A wild-type vs. knockout breast cancer cells should provide important insights into the regulation of intracellular iron homeostasis and cell metabolism in cells and tissues.
Non-invasive methods of tracking morphological cell changes are based on measurements of phase, which is proportional to the cell thickness and allows calculation of cell volume. Additionally, Raman micro-spectroscopy is widely used for the mapping of chemical composition within live biological samples, such as cells, organoids, and tissues. We have previously reported the use of Raman spectroscopy and Digital Holographic microscopy (DHM) to study cell death induced by methamphetamine treatment. Here, we have replaced DHM with another method that is capable of real-time high resolution phase reconstruction. Assembling or altering a system to make the measurements required to solve the Transport-of-Intensity Equation (TIE) is easier than implementing a DHM setup. For the full phase retrieval, TIE requires only the data collected in the focal plane and in two planes symmetrically positioned about the focus. Furthermore, TIE is robust to reduced spatial and temporal coherence. Since TIE can utilize incoherent sources of illumination, we implemented a TIE setup within an existing Raman microscope, which provided near simultaneous chemical composition and morphological cell data. This setup is well-suited to study another form of programmed cell death, ferroptosis, which is the main cause of tissue damage driven by iron overload and lipid peroxidation. Previously, only invasive cell biological assays were used to monitor the expression level and subcellular location of proteins known to bind iron or be involved in ferroptosis. In this work, our group applied Raman spectroscopic techniques to study MDA-MB-231 breast cancer cells treated with an activator and/or inhibitor of ferroptosis.
Transport of intensity (TIE) and digital holographic microscopy (DHM) are imaging techniques capable of real-time high resolution phase reconstructions. DHM is a widely used technique that provides phase maps through numerical reconstruction of light propagation of captured hologram intensities generated by interference between an object and a reference beam. TIE is a bright-field compatible technique that yields phase reconstructions through intensity measurements of a single object beam at symmetric planes about the focal plane. A TIE setup is simpler than DHM due to its non-interferometric nature and may yield a higher resolution reconstruction than DHM. Since TIE is a somewhat less-mature technique, we have developed a setup capable of both TIE and DHM measurements and simultaneously measured the volume changes of biological cells using both techniques. The setup is based on a modified bright-field microscope, with the addition of laser illumination for the DHM measurements. Live C6 glial cells were monitored as a hydrogen peroxide solution was introduced to the sample media to produce a visible and measurable decrease in cell volume through apoptosis. This decrease in volume was simultaneously measured by TIE and DHM, and the results were directly compared. Additionally, volume changes in C6 glial cells undergoing methamphetamine-induced apoptosis were tracked and compared.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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 μ's 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.
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.
PDT dose is the product of the photosensitizer concentration and the light fluence in target tissue. Although
existing systems are capable of measuring the light fluence in vivo, the concurrent measurement of
photosensitizer in the treated tissue so far has been lacking. We have developed and tested a new method to
simultaneously acquire light dosimetry and photosensitizer fluorescence data via the same isotropic detector,
employing treatment light as the excitation source. A dichroic beamsplitter is used to split light from the
isotropic detector into two fibers, one for light dosimetry, the other, after the 665 nm treatment light is
removed by a band-stop filter, to a spectrometer for fluorescence detection. The light fluence varies
significantly during treatment because of the source movement. The fluorescence signal is normalized by the
light fluence measured at treatment wavelength. We have shown that the absolute photosensitizer
concentration can be obtained by an optical properties correction factor and linear spectral fitting. Tissue
optical properties are determined using an absorption spectroscopy probe immediately before PDT at the
same sites. This novel method allows accurate real-time determination of delivered PDT dose using existing
isotropic detectors, and may lead to a considerable improvement of PDT treatment quality compared to the
currently employed systems. Preliminary data in patient studies is presented.
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.
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 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.
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