Optical anapole mode in nanostructured lithium niobate for enhancing second harmonic generation yang lia, zhijin huanga, zhan sui, huajiang chen, xinyue zhang, heyuan guan, wentao qiu, jiangli dong, wenguo zhu, jianhui yu, huihui lu, zhe chen a yang li and zhijin huang. The nonlinear imaging theory of second harmonic and third. Second harmonic generation shg microscopy uses samefrequency photons that interact with a noncentrosymmetric material and are combined to form new photons with twice the energy. Third harmonic generation thg microscopy is a labelfree scatter process that is. The aim of this research was to combine histology with new immune and histochemical tools. The swelling behavior of the alginate hydrogels was significantly higher under basic ph conditions. Higher harmonic generation microscopy of in vitro cultured mammal oocytes and embryos article pdf available in optics express 1615. Apr 18, 2014 second harmonic generation shg is a novel imaging technology that could provide optical biopsy during endoscopy with advantages over current technology.
Hematoxylin and eosinstained slides from colon biopsies normal, lowgrade dysplasia lgd, highgrade dysplasia hgd, and. In this article, we report the epi harmonic generation microscopic study of various abnormal enamel from the nature surface of human teeth. The utilization of multiharmonic imaging microscopy mhim to visualize. Membrane imaging by simultaneous second harmonic generation and twophoton microscopy l. Oct 17, 2017 enhanced image contrast in biological second harmonic imaging microscopy shim has previously been reported via quantitative assessments of forward to epigenerated signal intensity ratio and by. Structure sensitivity in thirdharmonic generation microscopy. In addition, results on the recent progress and impact shg microscopy has made in different areas of biology and medicine are presented. Second harmonic imaging microscopy shim is based on a nonlinear optical effect known as second harmonic generation shg. Second harmonic generation shg microscopy has emerged as a powerful modality for imaging fibrillar collagen in a diverse range of tissues. Polarized second harmonic generation optical microscopy for laserdirected assembly of zno nanowires ruiqing wang, fan wang, jing long, yufeng tao, linlin zhou, huange fu, yuncheng liu, binzhang jiao, leimin deng, and wei xiong. Here we show that lipid bodies are a major source of contrast in third harmonic generation thg microscopy of. May 08, 2014 also, the incident beam can be tuned to fit in the biological window of biological structures, which gives second harmonic generation microscopy a significant advantage over linear microscopy due to absorbance issues in the visible wavelength range. Higher harmonic generation microscopy hhgm, based on nonlinear multiphoton excitation, is a contrast mechanism for the structural and molecular imaging of native samples in cell culture and in fixed and live tissues, for both, three.
Optical second harmonic generation microscopy as a tool of. The nonlinear effect of shg and thg on the imaging properties of confocal microscopy. In vivo optical virtual biopsy of human oral mucosa with harmonic generation microscopy. In this article we compare the twophoton photoluminescence and second harmonic generation from single connected gold nanodimers. Second harmonic generation shg microscopy is an efficient technique to probe the 3d architecture of collagen, which is the main structural protein in mammals and determines the architecture of many tissues skin, cornea, artery, bone. Twophoton fluorescence and secondharmonic generation. Second harmonic generation shg imaging can be used.
Probing microtubules polarity in mitotic spindles in situ using. Osa epithird and second harmonic generation microscopic. Pdf secondharmonic generation shg microscopy has emerged as a powerful. Shg has the unique ability to evaluate the amount of extracellular matrix collagen protein and its alignment. Matcher2,3 enhanced image contrast in biological second harmonic imaging microscopy shim has previously.
Specific fluorescent probes were used to label the main biochemical components of the starchy fried matrix, namely starch and oil. Analyzing the particle sizedependent nonlinear optical spectra and performing excitation polarization resolved measurements using an experimental setup combining a femtosecond laser source with a parabolic mirror, we show that second harmonic generation and two. Request pdf higher harmonic generation microscopy higher harmonic generation, including second harmonic generation and third harmonic generation, leaves no energy deposition to the interacted. Pdf third harmonic generation microscopy of cells and tissue. Twophoton excited fluorescence microscopy in conventional confocal laser scanning microscopy, the fluorescence excitation source is a tightly focused continuouswave cw laser beam. An innovative methodology based on nondestructive observation by using harmonic generation microscopy is proposed for detection and location of starch granules and oil in a fried starchy matrix and topography analysis of food products. Imaging granularity of leukocytes with third harmonic. Nonlinear optical microscopy nlom is a well established tool for the interrogation of living biological specimen.
Shg signal originates from two excitation photons which interact with the material and are reconverted to form a new emitted photon with half of wavelength. Pdf second harmonic generation microscopy for quantitative. Third harmonic generation microscopy is used to make dynamical images of living systems for the first time. Membrane imaging by simultaneous secondharmonic generation and twophoton microscopy l.
Osa second harmonic generation imaging of collagen fibrils. Second harmonic generation microscopy in zebrafish. Multimodal twophoton imaging using a second harmonic. Total internal reflection fluorescence microscopy in single molecule nanobioscience. It is thus clinically important to diagnose the anomalies in tooth enamel structures in the early stage for prevention and treatment. For nonlinear microscopy, a specimen with a nonlinear optical crosssection will produce higher harmonic light emission under suf.
Holographic second harmonic generation microscopy infoscience. The nonlinear imaging theory of second harmonic generation shg and third harmonic generation thg in confocal microscopy is presented in this paper. A study on image quality in polarizationresolved second harmonic generation microscopy. Higher harmonic generation microscopy of in vitro cultured. These authors equally contributed to this work corresponding authors. Hybrid imaging methods are able to combine the specificity of collagen detection by shg with other narrow ranged fluorescent or biochemical markers, leading to higher efficiency of cancer diagnosis and grading. Stiffer gels consistently swelled to a lesser degree compared to softer gels for all conditions. Refractive index index of refraction the ratio of the speed of light in a vacuum to that in the imaging medium of a microscope. Collagen, as the most abundant protein in the human body, determines the unique physiological and optical properties of the connective tissues including cornea and sclera. Second harmonic generation microscopy reveals altered.
Bueno2 second harmonic generation shg microscopy represents a very powerful tool for tissue. Shg imaging revealed that corneal collagen fibrils are. Biological second and third harmonic generation microscopy. The corresponding merged thg purplefluorescence orange image is shown in fig.
Third harmonic generation thg from atherosclerotic plaques revealed. Multiphoton microscopy using laser sources in the midinfrared range. Second harmonic generation imaging distinguishes both high. Supplementary information pdf 504k article information. The second harmonic generation microscope shgm constructs images of intensity distributions of sh waves produced by the interaction of fundamental waves with a polar material. Polarized secondharmonic generation optical microscopy.
Membrane imaging by simultaneous secondharmonic generation. Quantum mechanically, higherharmonic generation involves the annihilation of n pump photons and generation of a photon. Multiphoton microscopy and second harmonic generation loci. We describe a novel second harmonic generation shg. Pdf study of apoptosis induction using fluorescent and. The multitude of imaging techniques two and threephoton excited fluorescence, second and third harmonic generation that encompass nonlinear microscopy allows the collection of structural, functional and chemical information at imaging depths of hundreds of microns within the. Second harmonic generation microscopy has emerged as a powerful new optical imaging modality.
Thirdharmonic generation microscopy with focusengineered. Twophoton and second harmonic microscopy in clinical and. In vivo imaging of myelin in the vertebrate central nervous system using third harmonic generation microscopy. Pdf we describe the principles and characteristics of. Optimization of secondharmonic generation microscopy article pdf available in micron 327. Higher harmonic generation microscopy for developmental biology chikuang sun,a, shiwei chu,a szuyu chen,a tsunghan tsai,a tzuming liu,a chungyung lin,b and huaijen tsaib a graduate institute of electrooptical engineering and department of electrical engineering, national taiwan university, taipei 10617, taiwan, roc b institute of molecular and cell biology, national taiwan university.
The project also involves the optimisation of the microscope and a quantitative study on the effects. The role of chiral local field enhancements below the. Excited by a 1230 nm femtosecond laser, thg signals were generated at a significantly higher level in neutrophils than other mononuclear cells, whereas signals in agranular lymphocytes were one. In second harmonic generation shg, laser light is focused on a sample to generate frequencydoubled light. Pdf optimization of secondharmonic generation microscopy. Higher harmonic generation microscopy of in vitro cultured mammal oocytes and embryos choshuen hsieh1, sheeuan chen 2, yenwei lee 1, yushih yang 2, and chikuang sun1, 3 1department of electrical engineering and graduate institute of photonics and optoelectronics, national taiwan university, taipei 10617, taiwan 2department of obstetrics and gynecology, national taiwan. A new mode of contrast in biological second harmonic. Shgm second harmonic generation microscopy acronymfinder. Tripling the maximum imaging depth with third harmonic generation microscopy murat yildirim,a nicholas durr,b,c and adela benyakara,c, athe university of texas at austin, department of mechanical engineering, 204 east dean keeton street, stop c2200, austin, texas 78712, united states bthe john hopkins university, department of biomedical engineering, 3400 north charles street, baltimore. Labelfree imaging of atherosclerotic plaques using thirdharmonic.
Caenorhabditis elegans, imaging, microscopy, shg, thg, tpef. Over the last few years, second harmonic generation shg microscopy. Mar 02, 2011 loss of myelin in the central nervous system cns leads to debilitating neurological deficits. Shg shares many of the advantageous features for microscopy of another more established nonlinear optical technique. Harmonic generation hg, also called multiple harmonic generation is a nonlinear optical process in which n photons with the same frequency interact with a nonlinear material, are combined, and generate a new photon with n times the energy of the initial photons equivalently, n times the frequency and the wavelength divided by n. In other cases, like secondharmonic imaging microscopy, only a tiny fraction of the. We combine this technique with a lowenergy kp model hamiltonian. In two photon fluorescence, a fluorophore absorbs two photons at a low. Higher harmonic generation microscopy springerlink.
Ultrastrong nonlinear optical processes and trigonal warping in. Shgm stands for second harmonic generation microscopy. The role of chiral local field enhancements below the resolution limit of second harmonic generation microscopy v. Pdf higher harmonic generation microscopy of in vitro. Chara plant rhizoids have been imaged, showing dynamic plant activity, and nonfading image characteristics even with continuous viewing, indicating prolonged viability under. Labelfree imaging of bone multiscale porosity and interfaces using.
This feature describes its chemical and physical principles and highlights current applications in disease diagnostics. Secondharmonic generation microscopy of collagenbearing. All materials possess thirdorder nonlinear susceptibility and higher order terms. This has led to a variety of powerful imaging tools, such as twophoton excited fluorescence microscopy tpm, second harmonic generation shg microscopy, and coherent antistokes raman scattering cars microscopy.
Interferometric second harmonic generation microscopy siavash yazdanfar, lily h. This dissertation is dedicated to the application of digital holography methods to second harmonic generation microscopy, and to the development of the resulting imaging technique. Higher harmonic generation microscopy for developmental biology article in journal of structural biology 1471. Two photon microscopy and second harmonic generation. Two photon microscopy and second harmonic generation leif gibb and david matthews introduction and background nonlinear microscopy has become an important tool for imaging biological samples such as neurons. Estimation of the second harmonic generation shg conversion efficiency. The extracellular matrix plays an important role in cellular balance, and collagen fibers are its most important component.
Estimation of helical angles of myosin and collagen by second harmonic generation imaging microscopy franc. When coupled with multiphoton fluorescence microscopy, shg can be used to image interactions between cells and the surrounding extracellular environment. We have developed this nonlinear optical microscope in order to make possible nondestructive, threedimensional 3d observations of various kinds of inorganic and organic materials. How is second harmonic generation microscopy abbreviated.
Pdf in vivo optical virtual biopsy of human oral mucosa. Shim has been established as a viable microscope imaging contrast mechanism for visualization of cell and tissue structure and function. Jul 04, 2000 in this study, we have developed a high performance microscopic system to perform second harmonic shimaging on a tooth. Secondharmonic generation is a nonlinear optical process in which two photons with the same. In biological tissue, type i and ii fibrillar collagen, microtubule assemblies including centrosomes and mitotic spindles and muscle myosin all endogenously.
Over the years, second harmonic generation shg microscopy has emerged as an effective tool in biology. Twophoton fluorescence and second harmonic generation imaging of collagen in human tissue based on multiphoton microscopy xingshan jiang,jiazhao zhong,yuchun liu,haibo yu,shuangmu zhuo, and jianxin chen institute of laser and optoelectronics technology, fujian provincial key laboratory for photonics technology. Tripling the maximum imaging depth with thirdharmonic. Because of its underlying physical origin, it is highly sensitive to the collagen fibrilfiber structure, and.
Highresolution optical imaging of myelin in the cns of animal models is limited by a lack of in vivo myelin labeling strategies. Nonlinear optical imaging of extracellular matrix proteins. An innovative methodology based on nondestructive observation by using harmonic generation microscopy is proposed for detection and location of starch granules and oil in a fried starchy matrix. Imaging of biological membranes in liquid institute for biophysics johannes kepler university of linz altenbergerstr. Rosin 1 and tillielouise hackett 1,2, 1 centre for heart lung innovation, university of british columbia, vancouver, bc v6z 1y6, canada. All these imaging modalities are compatible with twophoton excited. A study on image quality in polarizationresolved second. Investigating mechanisms of collagen thermal denaturation by high resolution second harmonic generation imaging. Twophoton and second harmonic microscopy in clinical and translational cancer research.
Shgm is defined as second harmonic generation microscopy rarely. Pdf the interaction of cells within their microenvironmental niche is fundamental to. Multimodal imaging of harmonophores and application of high. The outoffocus image degradation problem of oct also puts an impediment to its application to developmental biology studies. Investigating mechanisms of collagen thermal denaturation. Higher harmonic generation microscopy for developmental biology. We present a fast scanning transmissionmode confocal scanning laser microscope system based on the use of a second harmonic generation shg crystal for signal detection. Higher harmonic generation microscopy for developmental. Higher harmonic generation, including second harmonic generation and third harmonic generation, leaves no energy deposition to the interacted matters due to its virtuallevel transition characteristic, providing a truly noninvasive modality and is ideal for in vivo imaging of live specimens without any preparation. Enamel covers the tooth crown and is responsible for protecting the inner tissues of the teeth. The design and construction of a second harmonic generation. This resolving power is good enough for cellular imaging but still inferior to that of nonlinear microscopies including multiphoton fluorescence microscopy and higher harmonic generation microscopy.
This is a manuscript of an article published as boddapalli, anuraag, and kaitlin m. Second harmonic generation microscopy shg is a nondestructive imaging. May 27, 2015 these findings may offer key insights into the biologic underpinnings of refractory versus treatable pulmonary fibroses, and highlight the potential of second harmonic generation microscopy as a novel diagnostic tool for distinguishing between these clinical scenarios. Using third harmonic generation thg microscopy, we demonstrate that granularity differences of leukocytes can be revealed without a label.
The nonlinear harmonic generation is a function of the molecular structure. In particular, emerging techniques such as second harmonic generation shg microscopy can allow for the monitoring of intrinsic contrast, such as that from collagen, in live and fixed samples. Several laserscanning microscopy techniques have been developed in the last twenty years based on nonlinear optical phenomena. The resolving power of a microscope is the most important feature of the optical system. Tripling the maximum imaging depth with third harmonic generation microscopy murat yildirim,a nicholas durr,b,c and adela benyakara,c, athe university of texas at austin, department of mechanical engineering, 204 east dean keeton street, stop c2200, austin. Raman scattering cars, second harmonic generation shg or third harmonic generation thg are receiving considerable attention. Second harmonic generation microscopy of collagen organization in tunable, environmentally responsive alginate hydrogels. Quasar spectral detector at spectra between 380430, 499552, 579668nm and merge. Imaging lipid bodies in cells and tissues using third. Discussion of birefringence, brewsters angle, and various forms of polarized light.
Study of apoptosis induction using fluorescent and higher harmonic generation microscopy techniques in acartia tonsa nauplii exposed to chronic concentrations of nickel. Higher harmonicgeneration, including second harmonic generation and third harmonic generation, leaves no energy deposition to the interacted matters due to its virtuallevel transition characteristic, providing a truly noninvasive modality and is ideal for in vivo imaging of live specimens without any preparation. Precise determination of the crystallographic orientations. Interferometric second harmonic generation microscopy. Direct comparison of second harmonic generation and two.
In these microscopies, the signal is a coherent superposition of the. The quadratic intensity dependence of shg is exploited to preferentially reveal unscattered signal light and reject outoffocus scattered background. A second harmonic microscope obtains contrasts from variations in a specimens ability to generate second harmonic light from the. We exploit these advantages to characterize collagenbearing biological structures. Lipid bodies have an important role in energy storage and lipid regulation. Second harmonic generation microscopy of collagen organization in tunable. Second harmonic generation microscopy gets better contrast. The ultrastructure of collagen, which conventionally can only be resolved by electron microscopy, now can be probed by optical second harmonic generation shg imaging. Second harmonic generation shg has been developed in our laboratories as a highresolution nonlinear optical imaging microscopy for cellular membranes and intact tissues. Multiphoton microscopic imaging of collagen plays an important role in noninvasive diagnoses of human tissue. Second harmonic generation microscopy was originally developed alongside of incoherent nonlinear microscopy multiphoton excitation fluorescence, and largely.
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