Browse
Submit Data
Databases
API
Help

Dataset Information

65 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Utilizing Stimulated Raman Scattering Microscopy To Study Intracellular Distribution of Label-Free Ponatinib in Live Cells.

ABSTRACT: Stimulated Raman scattering (SRS) microscopy represents a powerful method for imaging label-free drug distribution with high resolution. SRS was applied to image label-free ponatinib with high sensitivity and specificity in live human chronic myeloid leukemia (CML) cell lines. This was achieved at biologically relevant, nanomolar concentrations, allowing determination of ponatinib uptake and sequestration into lysosomes during the development of acquired drug resistance and an improved understanding of target engagement.

SUBMITTER: Sepp K

PROVIDER: S-EPMC7073915 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Utilizing Stimulated Raman Scattering Microscopy To Study Intracellular Distribution of Label-Free Ponatinib in Live Cells.

Sepp Kristel K Lee Martin M Bluntzer Marie T J MTJ Helgason G Vignir GV Hulme Alison N AN Brunton Valerie G VG

Journal of medicinal chemistry 20191227 5

Stimulated Raman scattering (SRS) microscopy represents a powerful method for imaging label-free drug distribution with high resolution. SRS was applied to image label-free ponatinib with high sensitivity and specificity in live human chronic myeloid leukemia (CML) cell lines. This was achieved at biologically relevant, nanomolar concentrations, allowing determination of ponatinib uptake and sequestration into lysosomes during the development of acquired drug resistance and an improved understan ...[more]

PMID: 31829628

Similar Datasets

Label-free live-cell imaging of nucleic acids using stimulated Raman scattering microscopy.

Project description:Imaging of nucleic acids is important for studying cellular processes such as cell division and apoptosis. A noninvasive label-free technique is attractive. Raman spectroscopy provides rich chemical information based on specific vibrational peaks. However, the signal from spontaneous Raman scattering is weak and long integration times are required, which drastically limits the imaging speed when used for microscopy. Coherent Raman scattering techniques, comprising coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopy, overcome this problem by enhancing the signal level by up to five orders of magnitude. CARS microscopy suffers from a nonresonant background signal, which distorts Raman spectra and limits sensitivity. This makes CARS imaging of weak transitions in spectrally congested regions challenging. This is especially the case in the fingerprint region, where nucleic acids show characteristic peaks. The recently developed SRS microscopy is free from these limitations; excitation spectra are identical to those of spontaneous Raman and sensitivity is close to shot-noise limited. Herein we demonstrate the use of SRS imaging in the fingerprint region to map the distribution of nucleic acids in addition to proteins and lipids in single salivary gland cells of Drosophila larvae, and in single mammalian cells. This allows the imaging of DNA condensation associated with cell division and opens up possibilities of imaging such processes in vivo.

| S-EPMC3516876 | biostudies-literature

Label-free stimulated Raman scattering microscopy visualizes changes in intracellular morphology during human epidermal keratinocyte differentiation.

Project description:Epidermal keratinocyte (KC) differentiation, which involves the process from proliferation to cell death for shedding the outermost layer of skin, is crucial for the barrier function of skin. Therefore, in dermatology, it is important to elucidate the epidermal KC differentiation process to evaluate the symptom level of diseases and skin conditions. Previous dermatological studies used staining or labelling techniques for this purpose, but they have technological limitations for revealing the entire process of epidermal KC differentiation, especially when applied to humans. Here, we demonstrate label-free visualization of three-dimensional (3D) intracellular morphological changes of ex vivo human epidermis during epidermal KC differentiation using stimulated Raman scattering (SRS) microscopy. Specifically, we observed changes in nuclei during the initial enucleation process in which the nucleus is digested prior to flattening. Furthermore, we found holes left behind by improperly digested nuclei in the stratum corneum, suggesting abnormal differentiation. Our findings indicate the great potential of SRS microscopy for discrimination of the degree of epidermal KC differentiation.

| S-EPMC6715667 | biostudies-literature

Label-free DNA imaging in vivo with stimulated Raman scattering microscopy.

Project description:Label-free DNA imaging is highly desirable in biology and medicine to perform live imaging without affecting cell function and to obtain instant histological tissue examination during surgical procedures. Here we show a label-free DNA imaging method with stimulated Raman scattering (SRS) microscopy for visualization of the cell nuclei in live animals and intact fresh human tissues with subcellular resolution. Relying on the distinct Raman spectral features of the carbon-hydrogen bonds in DNA, the distribution of DNA is retrieved from the strong background of proteins and lipids by linear decomposition of SRS images at three optimally selected Raman shifts. Based on changes on DNA condensation in the nucleus, we were able to capture chromosome dynamics during cell division both in vitro and in vivo. We tracked mouse skin cell proliferation, induced by drug treatment, through in vivo counting of the mitotic rate. Furthermore, we demonstrated a label-free histology method for human skin cancer diagnosis that provides comparable results to other conventional tissue staining methods such as H&E. Our approach exhibits higher sensitivity than SRS imaging of DNA in the fingerprint spectral region. Compared with spontaneous Raman imaging of DNA, our approach is three orders of magnitude faster, allowing both chromatin dynamic studies and label-free optical histology in real time.

| S-EPMC4577158 | biostudies-literature

Label-free pathology by spectrally sliced femtosecond stimulated Raman scattering (SRS) microscopy.

Project description:Optical "virtual biopsy" is an attractive way to improve disease diagnosis and surgical guidance. Many optical microscopy techniques have been developed to provide diagnostic information without the need for tissue sectioning or staining. Among these techniques, label-free chemical imaging is the most desirable. Recently, it has been shown that narrowband, picosecond stimulated Raman scattering (SRS) can achieve comparable morphological contrast to hematoxylin and eosin staining (H&E staining), the 'gold standard' of pathology. However, to translate the technique from the bench to the bedside, optimal laser sources and parameters have yet to be identified. Here we describe an improvement to the narrowband SRS microscopy techniques for label-free tissue imaging. Through spectral slicing of broadband, femtosecond pulses, we are able to maintain the same protein/lipid contrast as narrowband SRS while achieving a higher signal-to-noise ratio (SNR). Our method draws upon the benefits of femtosecond pulses (e.g. higher peak power) while preserving those of picosecond pulses (e.g. adequate spectral resolution). We demonstrate this achievement through protein/lipid signal and contrast quantification of mouse brain tissue as a function of bandwidth, and comparison with numerical simulations. Further method validation is provided through imaging of additional mouse tissues: liver, kidney, and skin.

| S-EPMC5451135 | biostudies-literature

Label-free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy.

Project description:Label-free chemical contrast is highly desirable in biomedical imaging. Spontaneous Raman microscopy provides specific vibrational signatures of chemical bonds, but is often hindered by low sensitivity. Here we report a three-dimensional multiphoton vibrational imaging technique based on stimulated Raman scattering (SRS). The sensitivity of SRS imaging is significantly greater than that of spontaneous Raman microscopy, which is achieved by implementing high-frequency (megahertz) phase-sensitive detection. SRS microscopy has a major advantage over previous coherent Raman techniques in that it offers background-free and readily interpretable chemical contrast. We show a variety of biomedical applications, such as differentiating distributions of omega-3 fatty acids and saturated lipids in living cells, imaging of brain and skin tissues based on intrinsic lipid contrast, and monitoring drug delivery through the epidermis.

| S-EPMC3576036 | biostudies-literature

Rapid, label-free detection of brain tumors with stimulated Raman scattering microscopy.

Project description:Surgery is an essential component in the treatment of brain tumors. However, delineating tumor from normal brain remains a major challenge. We describe the use of stimulated Raman scattering (SRS) microscopy for differentiating healthy human and mouse brain tissue from tumor-infiltrated brain based on histoarchitectural and biochemical differences. Unlike traditional histopathology, SRS is a label-free technique that can be rapidly performed in situ. SRS microscopy was able to differentiate tumor from nonneoplastic tissue in an infiltrative human glioblastoma xenograft mouse model based on their different Raman spectra. We further demonstrated a correlation between SRS and hematoxylin and eosin microscopy for detection of glioma infiltration (? = 0.98). Finally, we applied SRS microscopy in vivo in mice during surgery to reveal tumor margins that were undetectable under standard operative conditions. By providing rapid intraoperative assessment of brain tissue, SRS microscopy may ultimately improve the safety and accuracy of surgeries where tumor boundaries are visually indistinct.

| S-EPMC3806096 | biostudies-literature

Label-Free Quantification of Apoptosis and Necrosis Using Stimulated Raman Scattering Microscopy.

Project description:Recombinant proteins are critical for modern therapeutics and diagnostics, with Chinese hamster ovary (CHO) cells serving as the primary production platform. However, environmental and chemical stressors in bioreactors often trigger cell death, particularly apoptosis, posing a significant challenge to recombinant protein manufacturing. Rapid, label-free methods to monitor cell death are essential for ensuring better production quality. Stimulated Raman scattering (SRS) microscopy offers a powerful, label-free approach to measure lipid and protein compositions in live cells. We demonstrate that SRS microscopy enables rapid and reagent-free analysis of apoptotic and necrotic transitions. Our results show that apoptotic cells exhibit higher protein concentrations, while necrotic cells show an opposite trend. To enhance analysis, we developed a quantitative single-cell analysis pipeline that extracts chemotypic and phenotypic signatures of apoptosis and necrosis, enabling the identification of subpopulations with varied responses to stressors or treatments. Furthermore, the cell death analysis was successfully generalized to other stressors and cell types. This study highlights SRS microscopy as a robust and non-invasive tool for rapid monitoring of live cell apoptotic and necrotic transitions. Our method and findings hold potential for improving quality control in CHO cell-based biopharmaceutical production and for evaluating cell death in diverse biological contexts.

| S-EPMC11908225 | biostudies-literature

Vibrational imaging of newly synthesized proteins in live cells by stimulated Raman scattering microscopy.

Project description:Synthesis of new proteins, a key step in the central dogma of molecular biology, has been a major biological process by which cells respond rapidly to environmental cues in both physiological and pathological conditions. However, the selective visualization of a newly synthesized proteome in living systems with subcellular resolution has proven to be rather challenging, despite the extensive efforts along the lines of fluorescence staining, autoradiography, and mass spectrometry. Herein, we report an imaging technique to visualize nascent proteins by harnessing the emerging stimulated Raman scattering (SRS) microscopy coupled with metabolic incorporation of deuterium-labeled amino acids. As a first demonstration, we imaged newly synthesized proteins in live mammalian cells with high spatial-temporal resolution without fixation or staining. Subcellular compartments with fast protein turnover in HeLa and HEK293T cells, and newly grown neurites in differentiating neuron-like N2A cells, are clearly identified via this imaging technique. Technically, incorporation of deuterium-labeled amino acids is minimally perturbative to live cells, whereas SRS imaging of exogenous carbon-deuterium bonds (C-D) in the cell-silent Raman region is highly sensitive, specific, and compatible with living systems. Moreover, coupled with label-free SRS imaging of the total proteome, our method can readily generate spatial maps of the quantitative ratio between new and total proteomes. Thus, this technique of nonlinear vibrational imaging of stable isotope incorporation will be a valuable tool to advance our understanding of the complex spatial and temporal dynamics of newly synthesized proteome in vivo.

| S-EPMC3710790 | biostudies-literature

In situ visualization of intracellular morphology of epidermal cells using stimulated Raman scattering microscopy.

Project description:Visualization of epidermal cells is important because the differentiation patterns of keratinocytes (KCs) are considered to be related to the functions and condition of skin. Optical microscopy has been widely used to investigate epidermal cells, but its applicability is still limited because of the need for sample fixation and staining. Here, we report our staining-free observation of epidermal cells in both tissue and culture by stimulated Raman scattering (SRS) microscopy that provides molecular vibrational contrast. SRS allowed us to observe a variety of cellular morphologies in skin tissue, including ladder-like structures in the spinous layer, enucleation of KCs in the granular layer, and three-dimensional cell column structures in the stratum corneum. We noticed that some cells in the spinous layer had a brighter signal in the cytoplasm than KCs. To examine the relevance of the observation of epidermal layers, we also observed cultured epidermal cells, including KCs at various differentiation stages, melanocytes, and Langerhans cell-like cells. Their SRS images also demonstrated various morphologies, suggesting that the morphological differences observed in tissue corresponded to the cell lineage. These results indicate the possible application of SRS microscopy to dermatological investigation of cell lineages and types in the epidermis by cellular-level analysis.

| S-EPMC5005979 | biostudies-literature

Label-free imaging of amyloid plaques in Alzheimer's disease with stimulated Raman scattering microscopy.

Project description:One of the key pathological features of Alzheimer's disease (AD) is the existence of extracellular deposition of amyloid plaques formed with misfolded amyloid-β (Aβ). The conformational change of proteins leads to enriched contents of β sheets, resulting in remarkable changes of vibrational spectra, especially the spectral shifts of the amide I mode. Here, we applied stimulated Raman scattering (SRS) microscopy to image amyloid plaques in the brain tissue of an AD mouse model. We have demonstrated the capability of SRS microscopy as a rapid, label-free imaging modality to differentiate misfolded from normal proteins based on the blue shift (~10 cm-1) of amide I SRS spectra. Furthermore, SRS imaging of Aβ plaques was verified by antibody staining of frozen thin sections and fluorescence imaging of fresh tissues. Our method may provide a new approach for studies of AD pathology, as well as other neurodegenerative diseases associated with protein misfolding.

| S-EPMC6239428 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data