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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
In oral and maxillofacial surgery, there is a need for tissue engineered constructs for dental implants, reconstructions due
to trauma, oral cancer or congenital defects. A non-invasive quality monitoring of the fabrication of tissue engineered
constructs during their production and implantation is a required component of any successful tissue engineering
technique. We demonstrate the design and application of a Raman spectroscopic probe for rapid and noninvasive
monitoring of Ex Vivo Produced Oral Mucosa Equivalent constructs (EVPOMEs). We conducted in vivo studies to
identify Raman spectroscopic failure indicators for EVPOMEs (already developed in vitro), and found that Raman
spectra of EVPOMEs exposed to thermal stress showed correlation of the band height ratio of CH2 deformation to phenylalanine ring breathing modes, providing a Raman metric to distinguish between viable and nonviable constructs.
This is the first step towards the ultimate goal to design a stand-alone system, which will be usable in a clinical setting,
as the data processing and analysis will be performed with minimal user intervention, based on already established and
tested Raman spectroscopic indicators for EVPOMEs.
Three-dimensional digital holographic microscopic phase imaging of objects that are thicker than the wavelength of the imaging light is ambiguous and results in phase wrapping. In recent years, several unwrapping methods that employed two or more wavelengths were introduced. These methods compare the phase information obtained from each of the wavelengths and extend the range of unambiguous height measurements. A straightforward dual-wavelength phase imaging method is presented which allows for a flexible tradeoff between the maximum height of the sample and the amount of noise the method can tolerate. For highly accurate phase measurements, phase unwrapping of objects with heights higher than the beat (synthetic) wavelength (i.e. the product of the original two wavelengths divided by their difference), can be achieved. Consequently, three-dimensional measurements of a wide variety of biological systems and microstructures become technically feasible. Additionally, an effective method of removing phase background curvature based on slowly varying polynomial fitting is proposed. This method allows accurate volume measurements of several small objects with the same image frame.
We show the application of near-infrared Raman Spectroscopy to in-vitro monitoring of the viability of tissue constructs
(EVPOMEs). During their two week production period EVPOME may encounter thermal, chemical or biochemical
stresses that could cause development to cease, rendering the affected constructs useless. We discuss the development of
a Raman spectroscopic technique to study EVPOMEs noninvasively, with the ultimate goal of applying it in-vivo. We
identify Raman spectroscopic failure indicators for EVPOMEs, which are stressed by temperature, and discuss the
implications of varying calcium concentration and pre-treatment of the human keratinocytes with Rapamycin. In
particular, Raman spectra show correlation of the peak height ratios of CH2 deformation to phenylalanine ring breathing,
providing a Raman metric to distinguish between viable and nonviable constructs. We also show the results of singular
value decomposition analysis, demonstrating the applicability of Raman spectroscopic technique to both distinguish
between stressed and non-stressed EVPOME constructs, as well as between EVPOMEs and bare AlloDerm® substrates,
on which the oral keratinocytes have been cultured. We also discuss complications arising from non-uniform thickness
of the AlloDerm® substrate and the cultured constructs, as well as sampling protocols used to detect local stress and
other problems that may be encountered in the constructs.
Coherent anti-Stokes Raman scattering (CARS) microscopy is a powerful technique to image the chemical composition
of complex samples in biophysics, biology and materials science. CARS is a four-wave mixing process. The application
of a spectrally narrow pump beam and a spectrally wide Stokes beam excites multiple Raman transitions, which are
probed by a probe beam. This generates a coherent directional CARS signal with several orders of magnitude higher
intensity relative to spontaneous Raman scattering. Recent advances in the development of ultrafast lasers, as well as
photonic crystal fibers (PCF), enable multiplex CARS. In this study, we employed two scanning imaging methods. In
one, the detection is performed by a photo-multiplier tube (PMT) attached to the spectrometer. The acquisition of a
series of images, while tuning the wavelengths between images, allows for subsequent reconstruction of spectra at each
image point. The second method detects CARS spectrum in each point by a cooled coupled charged detector (CCD)
camera. Coupled with point-by-point scanning, it allows for a hyperspectral microscopic imaging. We applied this
CARS imaging system to study biological samples such as oocytes.
KEYWORDS: Digital holography, Holography, 3D metrology, 3D image processing, Holograms, Phase imaging, Microscopy, Phase shift keying, Diffraction, 3D image reconstruction
Digital holography records the superposition of the object and reference waves. The subsequent reconstruction of both
amplitude and phase of the optical field is done by numerically propagating the optical field along the direction
perpendicular to the hologram plane in accordance with the laws of diffraction. Phase changes undergone by a light
wave passing through or reflecting from objects can be converted to the optical thickness or height measurements,
providing the three dimensional structural information about the object. Our dual wavelength phase imaging method
allows three dimensional measurements of a wide variety of biological systems and microstructures.
The scanning photon microscope technique is a method of microscopic image formation that employs a laser beam focused on a sample, while non-imaging detector receives the scattered light. The scans are achieved by means of a galvanometer based scanning mirror and a motorized micrometer. The system produces images analogous to the scanning electron microscopy with three-dimensional effects of shadowing and reflection. Compared to a conventional wide-field imaging system, the method allows for a greater ease of operation and flexibility, as the image quality is dependent upon the characteristics of the laser beam, rather than imaging optics. The image resolution on the order of a micron is demonstrated. A further gain in terms of resolution and the depth of focus by employment of Bessel rather than Gaussian beams is discussed. Additionally, we used a position-sensitive quadrant photodiode detector to highlight the overall spatial orientation of the imaged surface as well as its roughness. This concept can be useful in many areas, such as coherence imaging and fluorescence.
We describe the scanning photon microscopy (SPM), a new method of microscopic image formation that is analogous to the scanning electron microscopy but carried out entirely by optical means and therefore is without the cumbersome sample preparation procedures. A laser beam is focused and raster-scanned over the surface of an object by means of an a motorized micrometer. The light reflected from the object surface is collected by a detector that is placed at a finite angle with respect to the incident beam. The geometry of the system is such that it yields images reminiscent of the scanning electron microscopy with striking three-dimensional impression and the effects of shadowing and diffuse reflection. In the preliminary experiments several artificial and biological objects are imaged using this technique obtaining images with about 5 micron resolution and reasonable image quality. Because this is a scanned optical system, the optical quality requirement is significantly relaxed in comparison to conventional wide-field imaging system. We also consider the possibility of surpassing the wide-field system in terms of the resolution and depth of focus by use of Bessel optics. The simple concept can be extended in many directions such as fluorescence and nonlinear optical imaging.
We describe recent advances in the development of the original software for studying optics on the General Physics level. Two types of Windows software are reported: the guide-programs for simulation the basic optical experiments and the multichoice test-programs for teachers (to create tests) and for students (to check their knowledge). Application of guide-programs combined with image-files from CCD-camera is presented in two modes; with real equipment and simulation with empirical data- files. The testing system uses GRE approach and accompanied a lot of pictures with main optical circuits or charts. Some illustrations with real screen views for basic optical phenomena are presented.
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