Quantitative second-harmonic generation microscopy in collagen.
TL;DR: High-resolution second-harmonic generation microscopy was applied to confirm the hypothesis that regions in which collagen fibrils have the same orientation in rat tail tendon are likely to be less than approximately 1 microm in diameter.
Abstract: The second-harmonic signal in collagen, even in highly organized samples such as rat tail tendon fascicles, varies significantly with position. Previous studies suggest that this variability may be due to the parallel and antiparallel orientation of neighboring collagen fibrils. We applied high-resolution second-harmonic generation microscopy to confirm this hypothesis. Studies in which the focal spot diameter was varied from approximately 1 to approximately 6 microm strongly suggest that regions in which collagen fibrils have the same orientation in rat tail tendon are likely to be less than approximately 1 microm in diameter. These measurements required accurate determination of the focal spot size achieved by use of different microscope objectives; we developed a technique that uses second-harmonic generation in a quartz reference to measure the focal spot diameter directly. We also used the quartz reference to determine a lower limit (dXXX > 0.4 pm/V) for the magnitude of the second-order nonlinear susceptibility in collagen.
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TL;DR: It is proposed that quantifying collagen alignment is a viable, novel paradigm for the prediction of human breast cancer survival because of the strong statistical evidence for poor survival in patients with TACS and because the assessment can be performed in routine histopathological samples imaged via second harmonic generation or using picrosirius.
Abstract: Evidence for the potent influence of stromal organization and function on invasion and metastasis of breast tumors is ever growing. We have performed a rigorous examination of the relationship of a tumor-associated collagen signature-3 (TACS-3) to the long-term survival rate of human patients. TACS-3 is characterized by bundles of straightened and aligned collagen fibers that are oriented perpendicular to the tumor boundary. An evaluation of TACS-3 was performed in biopsied tissue sections from 196 patients by second harmonic generation imaging of the backscattered signal generated by collagen. Univariate analysis of a Cox proportional hazard model demonstrated that the presence of TACS-3 was associated with poor disease-specific and disease-free survival, resulting in hazard ratios between 3.0 and 3.9. Furthermore, TACS-3 was confirmed to be an independent prognostic indicator regardless of tumor grade and size, estrogen or progesterone receptor status, human epidermal growth factor receptor-2 status, node status, and tumor subtype. Interestingly, TACS-3 was positively correlated to expression of stromal syndecan-1, a receptor for several extracellular matrix proteins including collagens. Because of the strong statistical evidence for poor survival in patients with TACS, and because the assessment can be performed in routine histopathological samples imaged via second harmonic generation or using picrosirius, we propose that quantifying collagen alignment is a viable, novel paradigm for the prediction of human breast cancer survival.
1,020 citations
Journal Article•
TL;DR: It was found that chirality plays a significant role in the mechanism of contrast generation, and it is also shown that SHIM is highly sensitive to membrane potential, with a depolarization of 25 mV resulting in an approximately twofold loss of signal intensity.
Abstract: By adapting a laser scanning microscope with a titanium sapphire femtosecond pulsed laser and transmission optics, we are able to produce live cell images based on the nonlinear optical phenomenon of second harmonic generation (SHG). Second harmonic imaging (SHIM) is an ideal method for probing membranes of living cells because it offers the high resolution of nonlinear optical microscopy with the potential for near-total avoidance of photobleaching and phototoxicity. The technique has been implemented on three cell lines labeled with membrane-staining dyes that have large nonlinear optical coefficients. The images can be obtained within physiologically relevant time scales. Both achiral and chiral dyes were used to compare image formation for the case of single- and double-leaflet staining, and it was found that chirality plays a significant role in the mechanism of contrast generation. It is also shown that SHIM is highly sensitive to membrane potential, with a depolarization of 25 mV resulting in an approximately twofold loss of signal intensity.
456 citations
TL;DR: It is concluded that SHG and TPF can characterize differential microscopic features of the collagen hydrogel that are strongly correlated with bulk mechanical properties and may be a useful noninvasive tool to assess tissue mechanics.
346 citations
TL;DR: It is shown that SHG microscopy is highly specific for fibrillar collagens and that combined SHG and two-photon excited fluorescence (2PEF) imaging can provide simultaneous three-dimensional visualization of collagen synthesis and assembly sites in transgenic animal models expressing GFP constructs.
Abstract: We compare second harmonic generation (SHG) to histological and immunohistochemical techniques for the visualization and scoring of collagen in biological tissues. We show that SHG microscopy is highly specific for fibrillar collagens and that combined SHG and two-photon excited fluorescence (2PEF) imaging can provide simultaneous three-dimensional visualization of collagen synthesis and assembly sites in transgenic animal models expressing GFP constructs. Finally, we propose several scores for characterizing collagen accumulation based on SHG images and appropriate for different types of collagen distributions. We illustrate the sensitivity of these scores in a murine model of renal fibrosis using a morphological segmentation of the tissue based on endogenous 2PEF signals.
278 citations
TL;DR: A role for macrophages is revealed in collagen fibrillogenesis and in organization of the structure of terminal end buds in mice homozygous for a null mutation in the colony stimulating factor‐1 gene.
Abstract: Development of the ductal network in the mammary gland is dependent in part on the presence of macrophages. Here we utilize multi-photon microscopy and second harmonic generation to describe terminal end bud 3-dimensional structure and the organization of the surrounding collagen matrix. We have applied this approach to analyze the effect of macrophage deficiency on terminal end bud structure and collagen organization, using mice homozygous for a null mutation in the colony stimulating factor-1 gene (Csf1op/Csf1op). Primary terminal end buds have an oblong shape, with long collagen I fibers close to the neck of the terminal end bud and radiating upwards in the direction of growth. Around the terminal end buds, the amount of total collagen I detected by antibody staining was not affected by macrophage deficiency. However the amount of collagen I organized into long fibers, detected by second harmonic generation signal, was reduced in Csf1op/Csf1op mice. Macrophage deficiency also caused terminal end buds to be rounder and shorter. These studies reveal a role for macrophages in collagen fibrillogenesis and in organization of the structure of terminal end buds.
253 citations
Cites background from "Quantitative second-harmonic genera..."
...…from alpha triple helical matrices, including collagen type I, II, and III, acto-myosin, and tubulin when these structures are subjected to low-energy photons (Campagnola et al., 2001; Stoller et al., 2002a,b; Stoller et al., 2003; Mohler et al., 2003; Zipfel et al., 2003; Sahai et al., 2005)....
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...Another advantage of multi-photon microscopy over confocal microscopy is its ability to utilize a naturally occurring phenomenon known as second harmonic generation (SHG), which causes the resonant emission of polarized light from alpha triple helical matrices, including collagen type I, II, and III, acto-myosin, and tubulin when these structures are subjected to low-energy photons (Campagnola et al., 2001; Stoller et al., 2002a,b; Stoller et al., 2003; Mohler et al., 2003; Zipfel et al., 2003; Sahai et al., 2005)....
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References
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TL;DR: Owing to the high NIR penetration depth, non‐invasive optical biopsies can be obtained from patients and ex vivo tissue by morphological and functional fluorescence imaging of endogenous fluorophores such as NAD(P)H, flavin, lipofuscin, porphyrins, collagen and elastin.
Abstract: Summary Near infrared (NIR) multiphoton microscopy is becoming a novel optical tool of choice for fluorescence imaging with high spatial and temporal resolution, diagnostics, photochemistry and nanoprocessing within living cells and tissues. Three-dimensional fluorescence imaging based on non-resonant two-photon or three-photon fluorophor excitation requires light intensities in the range of MW cm 22 to GW cm 22 , which can be derived by diffraction limited focusing of continuous wave and pulsed NIR laser radiation. NIR lasers can be employed as the excitation source for multifluorophor multiphoton excitation and hence multicolour imaging. In combination with fluorescence in situ hybridization (FISH), this novel approach can be used for multi-gene detection (multiphoton multicolour FISH). Owing to the high NIR penetration depth, non-invasive optical biopsies can be obtained from patients and ex vivo tissue by morphological and functional fluorescence imaging of endogenous fluorophores such as NAD(P)H, flavin, lipofuscin, porphyrins, collagen and elastin. Recent botanical applications of multiphoton microscopy include depthresolved imaging of pigments (chlorophyll) and green fluorescent proteins as well as non-invasive fluorophore loading into single living plant cells. Non-destructive fluorescence imaging with multiphoton microscopes is limited to an optical window. Above certain intensities, multiphoton laser microscopy leads to impaired cellular reproduction, formation of giant cells, oxidative stress and apoptosis-like cell death. Major intracellular targets of photodamage in animal cells are mitochondria as well as the Golgi apparatus. The damage is most likely based on a two-photon excitation process rather than a onephoton or three-photon event. Picosecond and femtosecond laser microscopes therefore provide approximately the same safe relative optical window for two-photon vital cell studies. In labelled cells, additional phototoxic effects may occur via photodynamic action. This has been demonstrated for aminolevulinic acid-induced protoporphyrin IX and other porphyrin sensitizers in cells. When the light intensity in NIR microscopes is increased to TW cm 22 levels, highly localized optical breakdown and plasma formation do occur. These femtosecond NIR laser microscopes can also be used as novel ultraprecise nanosurgical tools with cut sizes between 100 nm and 300 nm. Using the versatile nanoscalpel, intracellular dissection of chromosomes within living cells can be performed without perturbing the outer cell membrane. Moreover, cells remain alive. Non-invasive NIR laser surgery within a living cell or within an organelle is therefore possible.
1,243 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: A broad range of excitation wavelengths are used to demonstrate that TPEF/SHG coregistration can easily be achieved in unstained tissues by using a simple backscattering geometry and the structural and molecular origin of the image-forming signal from the various tissue constituents was determined.
Abstract: Multiphoton microscopy relies on nonlinear light-matter interactions to provide contrast and optical sectioning capability for high-resolution imaging. Most multiphoton microscopy studies in biological systems have relied on two-photon excited fluorescence (TPEF) to produce images. With increasing applications of multiphoton microscopy to thick-tissue "intravital" imaging, second-harmonic generation (SHG) from structural proteins has emerged as a potentially important new contrast mechanism. However, SHG is typically detected in transmission mode, thus limiting TPEF/SHG coregistration and its practical utility for in vivo thick-tissue applications. In this study, we use a broad range of excitation wavelengths (730-880 nm) to demonstrate that TPEF/SHG coregistration can easily be achieved in unstained tissues by using a simple backscattering geometry. The combined TPEF/SHG technique was applied to imaging a three-dimensional organotypic tissue model (RAFT). The structural and molecular origin of the image-forming signal from the various tissue constituents was determined by simultaneous spectroscopic measurements and confirming immunofluorescence staining. Our results show that at shorter excitation wavelengths (<800 nm), the signal emitted from the extracellular matrix (ECM) is a combination of SHG and TPEF from collagen, whereas at longer excitation wavelengths the ECM signal is exclusively due to SHG. Endogenous cellular signals are consistent with TPEF spectra of cofactors NAD(P)H and FAD at all excitation wavelengths. The reflected SHG intensity follows a quadratic dependence on the excitation power, decays exponentially with depth, and exhibits a spectral dependence in accordance with previous theoretical studies. The use of SHG and TPEF in combination provides complementary information that allows noninvasive, spatially localized in vivo characterization of cell-ECM interactions in unstained thick tissues.
874 citations
TL;DR: In this paper, the refractive index, birefringence and their dispersions are analyzed accurately in a physically meaningful model of a dispersion equation having only five coefficients for the calcite and quartz crystals.
777 citations
TL;DR: In this article, the authors used the third harmonic generation near the focal point of a tightly focused beam to probe microscopical structures of transparent samples, which can resolve interfaces and inhomogeneities with axial resolution comparable to the confocal length of the beam.
Abstract: Third harmonic generation near the focal point of a tightly focused beam is used to probe microscopical structures of transparent samples. It is shown that this method can resolve interfaces and inhomogeneities with axial resolution comparable to the confocal length of the beam. Using 120 fs pulses at 1.5 μm, we were able to resolve interfaces with a resolution of 1.2 μm. Two-dimensional cross-sectional images have also been produced.
581 citations