The interaction of cells within their microenvironmental niche is fundamental to cell migration, positioning, growth and differentiation in order to form and maintain complex tissue organization and function. Second harmonic generation shg microscopy is a recently developed optical imaging technique capable of robust, powerful and unbiased labelfree direct detection of collagen fibril structure in tissue without the use of antibodies. Biological second and third harmonic generation microscopy. 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. Secondharmonic imaging microscopy shim is based on a nonlinear optical effect known as. 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. Highlights on selected microscopy techniques to study. Second harmonic generation microscopy is a novel technique. Higher harmonic generation microscopy springerlink. Third harmonic generation imaging for fast, labelfree. Highresolution plasma membraneselective imaging by second. Secondharmonic generation an overview sciencedirect topics. Therefore, various microscopy techniques, including multiphoton, lightsheet microscopy, and second harmonic generation, simplify the discovery of different types of internal functions in zebrafish. The former is a manual method only valid for a defined set of cover thickness values.
Third harmonic generation microscopy of cells and tissue organization. Within the point spread function of a tight focus, second. 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 fluorescence and secondharmonic generation. Pdf in vivo optical virtual biopsy of human oral mucosa. Two photon microscopy and second harmonic generation. This unit describes how higher harmonic generation microscopy hhgm is applied to detect native. In vivo optical virtual biopsy of human oral mucosa with harmonic generation microscopy. Secondorder nonlinear holographic microscopy for high speed, threedimensional imaging is demonstrated. Higher harmonic generation microscopy of in vitro cultured. Because of its underlying physical origin, it is highly sensitive to the collagen fibrilfiber structure, and. In this study, we show that second harmonic generation shg is a highresolution plasma membraneselective imaging technique that enables multifaceted investigations of the plasma membrane. Request pdf higher harmonic generation microscopy higher harmonicgeneration, including second harmonic generation and third harmonic generation, leaves no energy deposition to the interacted.
Optical microscopy and coherence tomography of cancer in. Second harmonic imaging microscopy shim is based on a nonlinear optical effect known as second harmonic generation shg. Oct 21, 2016 imaging of collagen with second harmonic generation microscopy. Pdf study of apoptosis induction using fluorescent and. Second harmonic generation microscopy has emerged as a powerful new optical imaging modality. Osa investigation of third harmonic generation confocal. Higher harmonic generation microscopy hgm can provide a noninvasive biopsy tool with excellent 3d spatial resolution because of its virtual transition and nonlinear characteristics. Third harmonic generation thg microscopy is a labelfree scatter process that is elicited by waterlipid and waterprotein interfaces, including intra and extracellular membranes, and. Imaging lipid bodies in cells and tissues using third. Labelfree second harmonic generation holographic microscopy of. In contrast to fluorescence imaging, shg specifically visualizes the plasma membrane at locations that are not attached to artificial substrates and allows.
Rebecca williams, biomedical engineering, cornell university because shg is a coherent process, some type of phase matching must occur between different nonlinear scattering elements in the focal volume in order for signal to be. In two photon fluorescence, a fluorophore absorbs two photons at a low. Second harmonic imaging microscopy shim involves illuminating non centrosymmetric structures with an ultrashort pulse of focused laser. The threedimensional 3d pointspread function psf is a key indicator for the practical imaging ability of thg confocal microscopy. All materials possess thirdorder nonlinear susceptibility and higherorder terms.
Second harmonic imaging microscopy of living cells pdf. Imaging lipid bodies in cells and tissues using third harmonic generation microscopy. Higher harmonic generation microscopy for developmental biology. The outoffocus image degradation problem of oct also puts an impediment to its application to developmental biology studies. Study of apoptosis induction using fluorescent and higher harmonic generation microscopy techniques in acartia tonsa nauplii exposed to chronic concentrations of nickel. Second harmonic generation imaging offers a complete guide to this optical modality, from basic principles, instrument. Osa higher harmonic generation microscopy of in vitro. May 01, 2016 we show that third harmonic generation thg microscopy provides labelfree, realtime images of histopathological quality. Second harmonic generation shg microscopy has shown great promise for imaging live cells and tissues, with applications in basic science, medical research, and tissue engineering. 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. It can also be shown that the higher the outofplane angle of the scaterrer, the. Second harmonic generation microscopy gets better contrast. A confocal configuration is added to the detection path in third harmonic generation thg microscopes for resolution improvement.
A remarkable corollary of this is that a much larger group of peptides and. We find that several key endogenous protein structures give rise to intense secondharmonic generation shgnonabsorptive frequency doubling of an excitation laser line. Higher harmonic generation microscope hgm, providing images with a 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. However, due to the coherent emission feature of thg, the 3d psf for thg confocal microscopy is still pending and has not been.
Higher harmonic generation microscopy for developmental. A new mode of contrast in biological second harmonic. 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. A new mode of contrast in biological second harmonic generation. 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. Characterization of oral precancerous lesions based on higher. High harmonic generation from atom clusters m aladi and i b foldeshigh harmonics generation at the interaction of an intense fs laser pulse with an overdense plasma layer a mihailescu, v stancalie and v paisrecent citations wavelet analysis of higher harmonics in tapping mode atomic force microscopy zhenyu wang et al. Choshuen hsieh, sheeuan chen, yenwei lee, yushih yang, and chikuang sun, higher harmonic generation microscopy of in vitro cultured mammal oocytes and.
Pdf when the energy density at the focal spot of a microscope is sufficiently large, nonlinear. Higher harmonic generation microscopy for developmental biology article in journal of structural biology 1471. Secondharmonic imaging microscopy shim on a laserscanning system proves, therefore, to be a powerful and unique tool for highresolution, high contrast. 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. Pdf optimization of secondharmonic generation microscopy. Secondharmonic imaging microscopy shim on a laserscanning system proves, therefore, to be a powerful and unique tool for highresolution, highcontrast, threedimensional studies of live cell and tissue architecture. 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 university. Jul 01, 2004 read higher harmonic generation microscopy for developmental biology, journal of structural biology on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips. Second harmonic generation microscopy reveals collagen fibres. Higher harmonics generation in tapping mode atomic force. In an attempt to explore the use of a potentially noninvasive imaging technique to assess and compare the alignment of human cervical collagen between pre and postmenopausal women, samples were analysed with shg microscopy. 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. Enhanced image contrast in biological second harmonic imaging microscopy shim has previously been reported via quantitative assessments of forward.
This feature describes its chemical and physical principles and highlights current applications in disease diagnostics. Virtual biopsy of rat tympanic membrane using higher harmonic. The second harmonic generation efficiencies for pulsed lasers can be higher than 30%, e. Aug 31, 2016 fully integrated reflectionmode photoacoustic, twophoton and second harmonic generation microscopy in vivo skip to main content thank you for visiting. Originally used in material sciences, the development of highpower lasers. Request pdf higher harmonic generation microscopy higher harmonic generation, including second harmonic generation and third harmonic generation, leaves no energy deposition to the interacted. Third harmonic generation thg microscopy is a labelfree scatter process. Secondharmonic imaging microscopy shim on a laserscanning system proves, therefore, to be a powerful and unique tool for highresolution, highcontrast.
The nonlinear harmonic generation is a function of the molecular structure. Imaging of biological membranes in liquid institute for biophysics johannes kepler university of linz altenbergerstr. Third harmonic generation microscopy of cells and tissue. Application of higher harmonic generation microscopy in. Fully integrated reflectionmode photoacoustic, twophoton. The second harmonic generation is widely used to extend the choice of wavelengths generated by lasers. Threedimensional highresolution secondharmonic 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. Shim has been established as a viable microscope imaging contrast mechanism for visualization of cell and tissue structure and function. For nonlinear microscopy, a specimen with a nonlinear optical crosssection will produce higher harmonic light emission under suf. Pdf secondthird harmonic generation microscopy researchgate. Thirdharmonic generation thg microscopy can be complementary to shg. Introduction all conventional optical microscopy modalities are restricted.
Second harmonic imaging microscopy shim involves illuminating noncentrosymmetric structures with an ultrashort pulse of focused laser. Second harmonic generation shg microscopy has emerged as a powerful modality for imaging fibrillar collagen in a diverse range of tissues. 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. Choshuen hsieh, sheeuan chen, yenwei lee, yushih yang, and chikuang sun, higher harmonic generation microscopy of in vitro cultured mammal oocytes and embryos, opt. Third harmonic generation microscopy of cells and tissue organization bettina weigelin1, gertjan bakker1 and peter friedl1,2,3, abstract the interaction of cells within their microenvironmental niche is fundamental to cell migration, positioning, growth and differentiation in order to form and maintain complex tissue organization and function.
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