Stimulated Raman scattering (SRS) microscopy enables label-free and quantitative imaging of active pharmaceutical ingredients within the skin, with superior chemical specificity and spatial and temporal resolution. Here, we present a method to study topical formulations on ex vivo human skin using two modalities, SRS and near-infrared light (NIR) transmission. NIR transmission is used to compensate for the SRS signal variance caused by differences in skin thickness and formulation properties. Optical co-registration of the two modalities enables recording the variance in each pixel. The developed method helps to evaluate the cutaneous pharmacokinetics of tretinoin from tretinoin-containing solution and cream formulations.
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.
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.
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.
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.
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