Pdf study of apoptosis induction using fluorescent and. Second harmonic generation shg has been developed in our laboratories as a highresolution nonlinear optical imaging microscopy for cellular membranes and intact tissues. 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. Higher harmonic generation microscopy springerlink. We exploit these advantages to characterize collagenbearing biological structures. In these microscopies, the signal is a coherent superposition of the. Quasar spectral detector at spectra between 380430, 499552, 579668nm and merge. We have developed this nonlinear optical microscope in order to make possible nondestructive, threedimensional 3d observations of various kinds of inorganic and organic materials. 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. Hematoxylin and eosinstained slides from colon biopsies normal, lowgrade dysplasia lgd, highgrade dysplasia hgd, and. Membrane imaging by simultaneous secondharmonic generation and twophoton microscopy l. This feature describes its chemical and physical principles and highlights current applications in disease diagnostics. Polarized secondharmonic generation optical microscopy. Over the last few years, second harmonic generation shg microscopy.
The design and construction of a second harmonic generation. 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. The outoffocus image degradation problem of oct also puts an impediment to its application to developmental biology studies. Second harmonic generation shg microscopy has emerged as a powerful modality for imaging fibrillar collagen in a diverse range of tissues. Structure sensitivity in thirdharmonic generation microscopy. Over the years, second harmonic generation shg microscopy has emerged as an effective tool in biology.
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. Third harmonic generation thg microscopy is a labelfree scatter process that is. Labelfree imaging of bone multiscale porosity and interfaces using. Shg imaging revealed that corneal collagen fibrils are. Secondharmonic generation is a nonlinear optical process in which two photons with the same.
Enamel covers the tooth crown and is responsible for protecting the inner tissues of the teeth. Membrane imaging by simultaneous second harmonic generation and twophoton microscopy l. Higher harmonic generation microscopy for developmental biology. Caenorhabditis elegans, imaging, microscopy, shg, thg, tpef. 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. Shgm is defined as second harmonic generation microscopy rarely. Multimodal imaging of harmonophores and application of high. Twophoton fluorescence and secondharmonic generation. Biological second and third harmonic generation microscopy. The aim of this research was to combine histology with new immune and histochemical tools. 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. 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. Multimodal twophoton imaging using a second harmonic. Pdf in vivo optical virtual biopsy of human oral mucosa.
Rosin 1 and tillielouise hackett 1,2, 1 centre for heart lung innovation, university of british columbia, vancouver, bc v6z 1y6, canada. How is second harmonic generation microscopy abbreviated. We describe a novel second harmonic generation shg. Multiphoton microscopy using laser sources in the midinfrared range. Third harmonic generation thg from atherosclerotic plaques revealed. The nonlinear harmonic generation is a function of the molecular structure. A second harmonic microscope obtains contrasts from variations in a specimens ability to generate second harmonic light from the. Membrane imaging by simultaneous secondharmonic generation. Pdf secondharmonic generation shg microscopy has emerged as a powerful. 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. Because of its underlying physical origin, it is highly sensitive to the collagen fibrilfiber structure, and. Study of apoptosis induction using fluorescent and higher harmonic generation microscopy techniques in acartia tonsa nauplii exposed to chronic concentrations of nickel.
Investigating mechanisms of collagen thermal denaturation. The role of chiral local field enhancements below the. Pdf we describe the principles and characteristics of. The project also involves the optimisation of the microscope and a quantitative study on the effects. Bueno2 second harmonic generation shg microscopy represents a very powerful tool for tissue. Apr 18, 2014 second harmonic generation shg is a novel imaging technology that could provide optical biopsy during endoscopy with advantages over current technology. Precise determination of the crystallographic orientations. Pdf second harmonic generation microscopy for quantitative. In vivo imaging of myelin in the vertebrate central nervous system using third harmonic generation microscopy. Nonlinear optical microscopy nlom is a well established tool for the interrogation of living biological specimen. 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. Twophoton and second harmonic microscopy in clinical and translational cancer research.
Discussion of birefringence, brewsters angle, and various forms of polarized light. In this article we compare the twophoton photoluminescence and second harmonic generation from single connected gold nanodimers. Labelfree imaging of atherosclerotic plaques using thirdharmonic. For nonlinear microscopy, a specimen with a nonlinear optical crosssection will produce higher harmonic light emission under suf. The nonlinear effect of shg and thg on the imaging properties of confocal microscopy. Second harmonic imaging microscopy shim is based on a nonlinear optical effect known as second harmonic generation shg. Here we show that lipid bodies are a major source of contrast in third harmonic generation thg microscopy of. The swelling behavior of the alginate hydrogels was significantly higher under basic ph conditions. In vivo optical virtual biopsy of human oral mucosa with harmonic generation microscopy.
Multiphoton microscopic imaging of collagen plays an important role in noninvasive diagnoses of human tissue. Shg has the unique ability to evaluate the amount of extracellular matrix collagen protein and its alignment. In second harmonic generation shg, laser light is focused on a sample to generate frequencydoubled light. Ultrastrong nonlinear optical processes and trigonal warping in. Holographic second harmonic generation microscopy infoscience. 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. Labelfree superresolution with coherent nonlinear structuredillumination microscopy mikko j huttunen1, aazad abbas1, jeremy upham1 and robert w boyd1,2 1department of physics, university of ottawa, ottawa, ontario k1n 6n5, canada 2the institute of optics and department of physics and astronomy, university of rochester, rochester, ny14627, united states of america. Second harmonic generation microscopy in zebrafish. Mar 02, 2011 loss of myelin in the central nervous system cns leads to debilitating neurological deficits. It is thus clinically important to diagnose the anomalies in tooth enamel structures in the early stage for prevention and treatment. Investigating mechanisms of collagen thermal denaturation by high resolution second harmonic generation imaging. These authors equally contributed to this work corresponding authors. 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. This is a manuscript of an article published as boddapalli, anuraag, and kaitlin m.
A new mode of contrast in biological second harmonic. Second harmonic generation microscopy reveals altered. Second harmonic generation microscopy of collagen organization in tunable. Quantum mechanically, higherharmonic generation involves the annihilation of n pump photons and generation of a photon. The nonlinear imaging theory of second harmonic and third. Twophoton excited fluorescence microscopy in conventional confocal laser scanning microscopy, the fluorescence excitation source is a tightly focused continuouswave cw laser beam. The quadratic intensity dependence of shg is exploited to preferentially reveal unscattered signal light and reject outoffocus scattered background. Osa epithird and second harmonic generation microscopic. The utilization of multiharmonic imaging microscopy mhim to visualize. Quantitative characterization of biological liquids for third. Higher harmonic generation microscopy of in vitro cultured.
Imaging granularity of leukocytes with third harmonic. Total internal reflection fluorescence microscopy in single molecule nanobioscience. The extracellular matrix plays an important role in cellular balance, and collagen fibers are its most important component. 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. Osa second harmonic generation imaging of collagen fibrils. Refractive index index of refraction the ratio of the speed of light in a vacuum to that in the imaging medium of a microscope.
Direct comparison of second harmonic generation and two. Shgm stands for second harmonic generation microscopy. A study on image quality in polarizationresolved second. 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. Specific fluorescent probes were used to label the main biochemical components of the starchy fried matrix, namely starch and oil. Second harmonic generation in skin and cornea laboratory.
In addition, results on the recent progress and impact shg microscopy has made in different areas of biology and medicine are presented. 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. Estimation of helical angles of myosin and collagen by second. The nonlinear imaging theory of second harmonic generation shg and third harmonic generation thg in confocal microscopy is presented in this paper. Two photon microscopy and second harmonic generation. 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. Second harmonic generation microscopy shg is a nondestructive imaging. 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. All materials possess thirdorder nonlinear susceptibility and higher order terms.
Higher harmonic generation microscopy of in vitro cultured mammal oocytes and embryos article pdf available in optics express 1615. In other cases, like secondharmonic imaging microscopy, only a tiny fraction of the. Interferometric second harmonic generation microscopy siavash yazdanfar, lily h. 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. Probing microtubules polarity in mitotic spindles in situ using. The high sensitivity of the system allows an acquisition rate of 300 secondsframe with a resolution at 512x512 pixels. 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. In this article, we report the epi harmonic generation microscopic study of various abnormal enamel from the nature surface of human teeth. Higher harmonic generation microscopy for developmental. Tripling the maximum imaging depth with thirdharmonic. Using third harmonic generation thg microscopy, we demonstrate that granularity differences of leukocytes can be revealed without a label.
Second harmonic generation shg imaging can be used. Supplementary information pdf 504k article information. 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. Pdf third harmonic generation microscopy of cells and tissue. 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. Shgm second harmonic generation microscopy acronymfinder. 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. Optical second harmonic generation microscopy as a tool 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.
Stiffer gels consistently swelled to a lesser degree compared to softer gels for all conditions. The resolving power of a microscope is the most important feature of the optical system. 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. Secondharmonic generation microscopy of collagenbearing. Estimation of the second harmonic generation shg conversion efficiency. Pdf optimization of secondharmonic generation microscopy. When coupled with multiphoton fluorescence microscopy, shg can be used to image interactions between cells and the surrounding extracellular environment. Thirdharmonic generation microscopy with focusengineered.
Second harmonic generation microscopy has emerged as a powerful new optical imaging modality. Interferometric second harmonic generation microscopy. Second harmonic generation microscopy gets better contrast. The corresponding merged thg purplefluorescence orange image is shown in fig. All these imaging modalities are compatible with twophoton excited. In two photon fluorescence, a fluorophore absorbs two photons at a low. 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.
We combine this technique with a lowenergy kp model hamiltonian. Shg shares many of the advantageous features for microscopy of another more established nonlinear optical technique. Matcher2,3 enhanced image contrast in biological second harmonic imaging microscopy shim has previously. Twophoton and second harmonic microscopy in clinical and. 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. Higher harmonic generation microscopy for developmental biology article in journal of structural biology 1471. Shim has been established as a viable microscope imaging contrast mechanism for visualization of cell and tissue structure and function. The role of chiral local field enhancements below the resolution limit of second harmonic generation microscopy v.
In biological tissue, type i and ii fibrillar collagen, microtubule assemblies including centrosomes and mitotic spindles and muscle myosin all endogenously. Pdf higher harmonic generation microscopy of in vitro. 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. Imaging of biological membranes in liquid institute for biophysics johannes kepler university of linz altenbergerstr. Multiphoton microscopy and second harmonic generation loci. Chara plant rhizoids have been imaged, showing dynamic plant activity, and nonfading image characteristics even with continuous viewing, indicating prolonged viability under. Nonlinear optical imaging of extracellular matrix proteins.
Lipid bodies have an important role in energy storage and lipid regulation. Highresolution optical imaging of myelin in the cns of animal models is limited by a lack of in vivo myelin labeling strategies. Imaging lipid bodies in cells and tissues using third. Second harmonic generation imaging distinguishes both high. A study on image quality in polarizationresolved second harmonic generation microscopy. Jul 04, 2000 in this study, we have developed a high performance microscopic system to perform second harmonic shimaging on a tooth. Raman scattering cars, secondharmonic generation shg or thirdharmonic generation thg are receiving considerable attention. 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. The ultrastructure of collagen, which conventionally can only be resolved by electron microscopy, now can be probed by optical second harmonic generation shg imaging. 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. Second harmonic generation microscopy of collagen organization in tunable, environmentally responsive alginate hydrogels. Several laserscanning microscopy techniques have been developed in the last twenty years based on nonlinear optical phenomena.
Second harmonic generation microscopy was originally developed alongside of incoherent nonlinear microscopy multiphoton excitation fluorescence, and largely. Estimation of helical angles of myosin and collagen by second harmonic generation imaging microscopy franc. 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. Third harmonic generation microscopy is used to make dynamical images of living systems for the first time. Request pdf higher harmonic generation microscopy higher harmonic generation, including second harmonic generation and third harmonic generation, leaves no energy deposition to the interacted. 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. Pdf the interaction of cells within their microenvironmental niche is fundamental to.