Second Harmonic Generation Imaging Microscopy: Applications to Diseases Diagnostics
TL;DR: Second Harmonic Generation microscopy has emerged as a powerful new optical imaging modality and its chemical and physical principles are described and current applications in disease diagnostics are highlighted.
Abstract: Second Harmonic Generation microscopy has emerged as a powerful new optical imaging modality. This Feature describes its chemical and physical principles and highlights current applications in disease diagnostics.
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TL;DR: This work discusses how second-harmonic generation microscopy can be used to obtain more structural information on the assembly of collagen in tissues than is possible by other microscopy techniques.
Abstract: Second-harmonic generation (SHG) microscopy has emerged as a powerful modality for imaging fibrillar collagen in a diverse range of tissues. Because of its underlying physical origin, it is highly sensitive to the collagen fibril/fiber structure, and, importantly, to changes that occur in diseases such as cancer, fibrosis and connective tissue disorders. We discuss how SHG can be used to obtain more structural information on the assembly of collagen in tissues than is possible by other microscopy techniques. We first provide an overview of the state of the art and the physical background of SHG microscopy, and then describe the optical modifications that need to be made to a laser-scanning microscope to enable the measurements. Crucial aspects for biomedical applications are the capabilities and limitations of the different experimental configurations. We estimate that the setup and calibration of the SHG instrument from its component parts will require 2-4 weeks, depending on the level of the user's experience.
739 citations
TL;DR: Fluorescence microscopes can both detect the fluorescence emitted from labeled molecules in biological samples as images or photometric data from which intensities and emission spectra can be deduced as discussed by the authors.
Abstract: Fluorescence microscopy provides an efficient and unique approach to study fixed and living cells because of its versatility, specificity, and high sensitivity. Fluorescence microscopes can both detect the fluorescence emitted from labeled molecules in biological samples as images or photometric data from which intensities and emission spectra can be deduced. By exploiting the characteristics of fluorescence, various techniques have been developed that enable the visualization and analysis of complex dynamic events in cells, organelles, and sub-organelle components within the biological specimen. The techniques described here are fluorescence recovery after photobleaching (FRAP), the related fluorescence loss in photobleaching (FLIP), fluorescence localization after photobleaching (FLAP), Forster or fluorescence resonance energy transfer (FRET) and the different ways how to measure FRET, such as acceptor bleaching, sensitized emission, polarization anisotropy, and fluorescence lifetime imaging microscopy (FLIM). First, a brief introduction into the mechanisms underlying fluorescence as a physical phenomenon and fluorescence, confocal, and multiphoton microscopy is given. Subsequently, these advanced microscopy techniques are introduced in more detail, with a description of how these techniques are performed, what needs to be considered, and what practical advantages they can bring to cell biological research.
429 citations
TL;DR: This Review highlights the potential of UCNPs for labeling and encoding biomolecules, microspheres, and even whole cells and lays the foundation for highly multiplexed analyte detection.
Abstract: Photon-upconverting nanoparticles (UCNPs) are lanthanide-doped nanocrystals that emit visible light under near-infrared excitation (anti-Stokes emission). This unique optical property precludes background fluorescence and light scattering from biological materials. The emission of multiple and narrow emission lines is an additional hallmark of UCNPs that opens up new avenues for optical encoding. Distinct emission signatures can be obtained if the multiple emission of UCNPs is tuned by their dopant composition or by surface modification with dyes. Tuning the intensity of only one of the multiple emission lines and using another one as a constant reference signal enables the design of ratiometric codes that are resistant to fluctuations in absolute signal intensities. Combining several UCNPs each displaying a distinct set of emission lines expands the coding capacity exponentially and lays the foundation for highly multiplexed analyte detection. This Review highlights the potential of UCNPs for labeling and encoding biomolecules, microspheres, and even whole cells.
385 citations
TL;DR: A review of the key advances that have occurred in the past several years in the field of single cell optical imaging to highlight the types of findings that are possible at the nexus of microscopy, nanoprobes, and live cells.
Abstract: In his 1665 treatise, Micrographia, Robert Hooke described the many observations he had made using a microscope, including compartment-like structures in cork samples that he termed ‘cells’.1 In the three and a half centuries since Hooke’s day, both the microscope and our understanding of the cell have been vastly improved upon, and the current outlook suggests that the symbiotic relationship between the microscope and the cell will continue to flourish into the foreseeable future. The cell is a basic yet complicated ‘unit’ of interest to biology, just as the atom is to chemists. Ultimately, scientists want to ‘see to believe’ when it comes to an explanation of the complex inner workings of cells, but therein lies a complication. Seeing is not always a possibility in biological systems. Size, speed, sensitivity, and additional concerns plague the microscopist who wants to peek inside of a cell. Enter a variety of molecular and nanoparticle probes that are capable of tagging and pinpointing the location of biological components that would otherwise be invisible under the microscope. Advances in laser, camera, and imaging processing technologies have also played a crucial role in the burgeoning field of single cell imaging, because they have brought into view the fast processes that would normally escape the human eye.
The purpose of this review is to highlight the key advances that have occurred in the past several years in the field of single cell optical imaging. It is not our intent to provide a comprehensive review of the types of experiments or the areas of cell research that are ongoing. Reviews with a distinctly biological flavor have been published recently, and these alternative reviews focus on specific details of the cell and the processes that occur within.2-7 Likewise, exceptional review papers that have discussed the full spectrum of nanoparticle probes and their properties have appeared recently.6-12 This review is designed to give an overview of the tools that are being specifically used to accomplish single cell imaging. As such, much of our emphasis in the first several sections of this paper is on imaging platforms, with a focus on design details that are important to single cell imaging experiments. Next we emphasize specific imaging experiments that highlight the types of findings that are possible at the nexus of microscopy, nanoprobes, and live cells. Particular attention is paid to the emerging orientation and rotational tracking of single probes linked to mechanistic functions and differentiated structures of biological interest. Finally, we provide a brief, yet rather complete, summary of single cell manipulation techniques.
249 citations
TL;DR: The results suggest that the passive human LV myocardium under quasi-static and dynamic multiaxial loadings is a nonlinear, anisotropic (orthotropic), viscoelastic and history-dependent soft biological material undergoing large deformations.
207 citations
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TL;DR: Hydrogels are an appealing scaffold material because they are structurally similar to the extracellular matrix of many tissues, can often be processed under relatively mild conditions, and may be delivered in a minimally invasive manner.
4,573 citations
TL;DR: Three tumor-associated collagen signatures (TACS) are observed and defined that provide novel markers to locate and characterize tumors and should provide indications that a tumor is, or could become, invasive, and may serve as part of a strategy to help identify and characterize breast tumors in animal and human tissues.
Abstract: Stromal-epithelial interactions are of particular significance in breast tissue as misregulation of these interactions can promote tumorigenesis and invasion. Moreover, collagen-dense breast tissue increases the risk of breast carcinoma, although the relationship between collagen density and tumorigenesis is not well understood. As little is known about epithelial-stromal interactions in vivo, it is necessary to visualize the stroma surrounding normal epithelium and mammary tumors in intact tissues to better understand how matrix organization, density, and composition affect tumor formation and progression. Epithelial-stromal interactions in normal mammary glands, mammary tumors, and tumor explants in three-dimensional culture were studied with histology, electron microscopy, and nonlinear optical imaging methodologies. Imaging of the tumor-stromal interface in live tumor tissue ex vivo was performed with multiphoton laser-scanning microscopy (MPLSM) to generate multiphoton excitation (MPE) of endogenous fluorophores and second harmonic generation (SHG) to image stromal collagen. We used both laser-scanning multiphoton and second harmonic generation microscopy to determine the organization of specific collagen structures around ducts and tumors in intact, unfixed and unsectioned mammary glands. Local alterations in collagen density were clearly seen, allowing us to obtain three-dimensional information regarding the organization of the mammary stroma, such as radiating collagen fibers that could not have been obtained using classical histological techniques. Moreover, we observed and defined three tumor-associated collagen signatures (TACS) that provide novel markers to locate and characterize tumors. In particular, local cell invasion was found predominantly to be oriented along certain aligned collagen fibers, suggesting that radial alignment of collagen fibers relative to tumors facilitates invasion. Consistent with this observation, primary tumor explants cultured in a randomly organized collagen matrix realigned the collagen fibers, allowing individual tumor cells to migrate out along radially aligned fibers. The presentation of these tumor-associated collagen signatures allowed us to identify pre-palpable tumors and see cells at the tumor-stromal boundary invading into the stroma along radially aligned collagen fibers. As such, TACS should provide indications that a tumor is, or could become, invasive, and may serve as part of a strategy to help identify and characterize breast tumors in animal and human tissues.
1,524 citations
TL;DR: This study provides the first data causally linking increased stromal collagen to mammary tumor formation and metastasis, and demonstrates that fundamental differences arise and persist in epithelial tumor cells that progressed within collagen-dense microenvironments.
Abstract: Mammographically dense breast tissue is one of the greatest risk factors for developing breast carcinoma. Despite the strong clinical correlation, breast density has not been causally linked to tumorigenesis, largely because no animal model has existed for studying breast tissue density. Importantly, regions of high breast density are associated with increased stromal collagen. Thus, the influence of the extracellular matrix on breast carcinoma development and the underlying molecular mechanisms are not understood. To study the effects of collagen density on mammary tumor formation and progression, we utilized a bi-transgenic tumor model with increased stromal collagen in mouse mammary tissue. Imaging of the tumors and tumor-stromal interface in live tumor tissue was performed with multiphoton laser-scanning microscopy to generate multiphoton excitation and spectrally resolved fluorescent lifetimes of endogenous fluorophores. Second harmonic generation was utilized to image stromal collagen. Herein we demonstrate that increased stromal collagen in mouse mammary tissue significantly increases tumor formation approximately three-fold (p < 0.00001) and results in a significantly more invasive phenotype with approximately three times more lung metastasis (p < 0.05). Furthermore, the increased invasive phenotype of tumor cells that arose within collagen-dense mammary tissues remains after tumor explants are cultured within reconstituted three-dimensional collagen gels. To better understand this behavior we imaged live tumors using nonlinear optical imaging approaches to demonstrate that local invasion is facilitated by stromal collagen re-organization and that this behavior is significantly increased in collagen-dense tissues. In addition, using multiphoton fluorescence and spectral lifetime imaging we identify a metabolic signature for flavin adenine dinucleotide, with increased fluorescent intensity and lifetime, in invading metastatic cells. This study provides the first data causally linking increased stromal collagen to mammary tumor formation and metastasis, and demonstrates that fundamental differences arise and persist in epithelial tumor cells that progressed within collagen-dense microenvironments. Furthermore, the imaging techniques and signature identified in this work may provide useful diagnostic tools to rapidly assess fresh tissue biopsies.
1,205 citations
TL;DR: Second-harmonic imaging microscopy (SHIM) on a laser-scanning system proves, therefore, to be a powerful and unique tool for high-resolution, high-contrast, three-dimensional studies of live cell and tissue architecture.
936 citations
TL;DR: Fundamental principles governing SHG phase matching with the tightly focusing optics used in microscopy are discussed, which are crucial to the design and development of forthcoming diagnostic and research tools.
862 citations