Shg and thg are nonlinear optical microscopic techniques that do not require labeling of tissue to generate 3d images with intrinsic depth. Highresolution structures of proteins reveal insights into function and enable rational drug design. Second and third harmonic generation microscopy visualizes. Secondorder nonlinear holographic microscopy for highspeed, threedimensional imaging is demonstrated. It can also be shown that the higher the outofplane angle of the scaterrer, the. First, multiphoton processes are excited only at the focal spot where the photon flux density is high enough for the nonlinear process to occur. Matcher2,3 enhanced image contrast in biological second harmonic imaging microscopy shim has previously. 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. For instance, second harmonic generation shg allows markerfree identification of cell. Adaptive harmonic generation microscopy of mammalian embryos.
Enhancing resolution and contrast in secondharmonic generation microscopy using an advanced maximum likelihood estimation restoration method amayandi sivaguru, bmohammad m. Second harmonic generation shg is a coherent optical process of radiation of dipoles in the material, dependent on the second term of the expansion of polarization. Fully integrated reflectionmode photoacoustic, twophoton. Third harmonic generation microscopy of cells and tissue.
Second harmonic generation microscopy has emerged as a powerful new optical imaging modality. Higher harmonic generation microscopy springerlink. Third harmonic generation imaging for fast, labelfree. Estimation of helical angles of myosin and collagen by second. Multiphoton microscopy and second harmonic generation loci. All these imaging modalities are compatible with twophoton excited. In this context, third harmonic generation thg microscopy is a promising tool because of its capacity to visualize almost the full morphology of tissue. Modern shg instrumentation typically has two primary components. 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.
Class 5 photonics offer powerful and highperformance femtosecond lasers based on optical parametric chirped pulse amplification opcpa suitable for higher harmonic generation hhg. Pdf we describe the principles and characteristics of. Imaging with secondharmonic generation nanoparticles. Here we demonstrate the first photomultiplierbased realtime secondharmonicgeneration microscopy taking advantage of the strongly. Aberration correction is performed to compensate both system and specimeninduced aberrations by using an efficient optimization routine based upon zernike polynomial modes. Shgm is a variant of two photon 2p microscopy that can detect the fibrillar collagens fcs without exogenous labels. Fluorescence emission is nearly simultaneous with the absorption of the.
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. We have acquired holograms with acquisition times as short as 10 ms, limited by the switching speed of our shutter. Thirdharmonic generation thg microscopy can be complementary to shg. Third harmonic generation microscopy of gan abstract. Third harmonic thg microscopy laboratory for optics. Here, we demonstrate that second harmonic generation shg and multiphoton fluorescence microscopy can quantitatively differentiate normal and ipf human tissues. Rebecca williams, biomedical engineering, cornell university because shg is a coherent process, some type of phase matching must occur between different nonlinear. Higher harmonic generation microscopy for developmental. 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. Shg has the unique ability to evaluate the amount of extracellular matrix collagen protein and its alignment.
To explore this question, we used second harmonic generation shg microscopy shgm to compare the matrix of uip and cop to that of healthy lung tissue. Thirdharmonic generation microscopy by use of a compact, femtosecond fiber laser source. Pdf when the energy density at the focal spot of a microscope is sufficiently large, nonlinear optical effects such as. Pdf secondthird harmonic generation microscopy shakil. A strong emphasis has been on developing these methods as.
Polarizationresolved second harmonic microscopy in collagenous. Shim has been established as a viable microscope imaging contrast mechanism for visualization of cell and tissue structure and function. 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 is a novel imaging technology that could provide optical biopsy during endoscopy with advantages over current technology. Biological second and third harmonic generation microscopy. Study of apoptosis induction using fluorescent and higher harmonic generation microscopy techniques in acartia tonsa nauplii exposed to chronic concentrations of nickel.
We demonstrate the first use, to our knowledge, of a compact, diodepumped, femtosecond fiber laser for thirdharmonic generation thg microscopy. We have developed this nonlinear optical microscope in order to make possible nondestructive, threedimensional 3d observations of various kinds of inorganic and organic materials. Shg microscopy assessment of hs with pearson correlation of 0. Rp photonics encyclopedia high harmonic generation, hhg. Multiphoton microscopy has become a standard method for noninvasive imaging of thick specimens with subcellular resolution. Both the second and third harmonic intensity signals are used as the optimization metric. Realtime secondharmonicgeneration microscopy based on a 2ghz repetition rate ti.
Second harmonic generation shg microscopy describes a technique whereby samefrequency photons interact with a noncentrosymmetric material and. Causes, effects and minimiation apers to understand how distortion is transferred from current harmonics injection into harmonic voltage distortion, its necessary to indroduce the concept of point of common coupling pcc. Second harmonic generation shg microscopy is regarded nowadays as a very useful and powerful tool for characterizing biological tissues1,2. Thirdharmonic generation microscopy with focusengineered. Hematoxylin and eosinstained slides from colon biopsies normal, lowgrade dysplasia lgd, highgrade dysplasia hgd. In biological tissue, type i and ii fibrillar collagen, microtubule assemblies including centrosomes and mitotic spindles and muscle myosin all endogenously produce a shg signal without exogenous probes.
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. Second harmonic generation shg imaging microscopy has recently appeared to be a power ful tool to image intrinsic subcellular signals from endogeneous proteins such as microtubule, myosin and collagen in living tissues 1, 2, 3. 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. Applications to diseases diagnostics paul campagnola his research interests are in the development of nonlinear optical microscopy techniques for cellular and tissue imaging, as well as the development of 3d nanomicrofabrication methods for biomedical applications. The fibrillar collagen in sinusoids of normal livers could be observed. Osa thirdharmonic generation microscopy by use of a. Barton auniversity of arizona, biomedical engineering, 1657 e. Sensitive and selective detection of protein crystals second harmonic generation microscopy is being explored for sensitive detection and characterization or protein crystals. It has tremendous implications for intravital shg microscopy. Realtime secondharmonicgeneration microscopy based on a. Light microscopy is the method of choice for intravital studies of small animals. In particular, thirdharmonic generation thg microscopy detects interfaces and optical heterogeneities and provides 3d structural images of unstained biological. Here, as a step towards this purpose, the potential of second and third harmonic generation shg, thg microscopy is explored.
Studying metalorganic frameworks using second harmonic. Second harmonic generation microscopy gets better contrast. Thg is a nonlinear optical process that relies on spatial variations of the thirdorder nonlinear susceptibility. The design and construction of a second harmonic generation. A study on image quality in polarizationresolved second. Multiphoton microscopy, including shg, produces highresolution, threedimensional pictures of tissue with minimal damage to living cells. Second harmonic imaging microscopy of living cells pdf. 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. Although shgm seems to provide higher resolution under the same excitation wavelength, the resolution is still limited by the diffraction limit. Second harmonic generation imaging using a confocal microscope second harmonic generation shg can generate a wave with twice the optical frequency. Enhancing resolution and contrast in secondharmonic.
Analysis of secondharmonicgeneration microscopy in a mouse model of ovarian carcinoma jennifer m. Second harmonic generation imaging using a confocal. During shg experiments of gan using a femtosecond cr. Hematoxylin and eosinstained slides from colon biopsies normal, lowgrade dysplasia lgd, highgrade dysplasia hgd, and. Combined with its nonlinearity, higher harmonic generation microscopy provides excellent threedimensional 3d sectioning capability, offering new insights into the studies of embryonic morphological changes and complex developmental processes. Raman scattering cars, secondharmonic generation shg or thirdharmonic generation thg are receiving considerable attention. May 12, 2009 second harmonic generation microscopy gets better contrast researchers use a trick of light to increase the image contrast in second chris lee may 12, 2009 2. Third harmonic generation microscopy is used to make dynamical images of living systems for the first time.
Secondharmonic imaging microscopy shim is based on a nonlinear optical effect known as. Labelfree imaging of bone multiscale porosity and interfaces using. When the energy density at the focal spot of a microscope is sufficiently large, nonlinear optical effects such as, harmonic generation, sumfrequency generation, coherent raman scattering, parametric oscillations, and multiphoton fluorescence can. Second harmonic generation in nonlinear optical crystal. The generation rate for each harmonic can therefore be calculated which, along with the predicted photon polarization and harmonic spectral distributions, agree quite well with experimental data. Second harmonic generation shg has been developed in our laboratories as a highresolution nonlinear optical imaging microscopy for cellular membranes and intact tissues. For shg analysis, we developed a classifier based on wavelet transforms, principle component analysis, and a knearestneighbor algorithm to classify the specific alterations of the. Two photon microscopy and second harmonic generation. Highharmonic generation by field enhanced femtosecond. Electrooptic mechanism of membrane potential sensitivity jiang jiang, kenneth b. May 01, 2011 second harmonic generation imaging microscopy.
Hhg data we now discuss some real experimental high harmonic spectra that have been observed in experiments. Second harmonic generation shg microscopy is a recently developed optical imaging technique capable of robust and powerful labelfree detection of collagen fibril structure in tissue. Pdf study of apoptosis induction using fluorescent and. Apr 18, 2014 second harmonic generation shg is a novel imaging technology that could provide optical biopsy during endoscopy with advantages over current technology. Eisenthal,y and rafael yuste howard hughes medical institute, department of biological sciences, andydepartment of chemistry, columbia university, new york. Second harmonic generation microscopy reveals altered.
Tripling the maximum imaging depth with thirdharmonic. Shg shares many of the advantageous features for microscopy of another more established nonlinear optical technique. Analysis of secondharmonicgeneration microscopy in a. Secondharmonic generation microscopy analysis reveals proteoglycan decorin is necessary for proper collagen organization in prostate. Realtime secondharmonicgeneration microscopy based on. Because of its underlying physical origin, it is highly sensitive to the collagen fibrilfiber structure, and, importantly, to changes that occur in diseases such as cancer, fibrosis and connective tissue. Harmonic generation microscopy hgm was recently applied to image biological samples.
Pdf thirdharmonic generation microscopy by use of a. Since thg exists in centrosymmetric structures it can be an excellent label for the mapping of the crystal property itself. Commonly used methods, histological staining followed by either semiquantitative or computerized image analysis, have limited sensitivity, accuracy, and operatordependent variations. Introduction when organic or inorganic specimens absorb and subsequently reradiate light, the process is typically a result of fluorescence or phosphorescence. We have recently proposed and demonstrated a novel nonlinear scanning laser microscope that uses thirdharmonic generation thg to characterize transparent specimen 10. Secondharmonic imaging microscopy of living cells drs. 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. Harmonic generation microscopy is a noninvasive optical technique suitable for visualizing biological structures in vivo with threedimensional 3d sectioning, endogenous contrast and low outof. Second harmonic generation third harmonic generation scanning microscopy higher harmonic generation multi harmonic generation. A material with a higher linear susceptibility, has a greater ability to polarize in response to the. Studying metalorganic frameworks using second harmonic generation microscopy nanowerk news linkam scientific instruments report on the use of their thms600 temperature controlled stage in the study of metalorganic frameworks using second harmonic generation microscopy at the university of leuven. The outoffocus image degradation problem of oct also puts an impediment to its application to developmental biology studies. The use of harmonic generation allows image formation of endogenous features in biological samples such as muscle tissue.
The first image is a block diagram noting the input to and output from the nonlinear optical material. Laser scanning thirdharmonicgeneration microscopy in biology. 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. Our resident insectivore, mole, has started a new series the corona files. The fibrilforming collagens include collagen types 1 3, 5. The second image is an energylevel diagram of the phenomenon. A new mode of contrast in biological second harmonic. Second harmonic imaging microscopy shim is based on a nonlinear optical effect known as second harmonic generation shg. Aug 31, 2016 among all these optical microscopic technologies, multiphoton fluorescence microscopy and second harmonic generation shg microscopy are capable of providing specific types of molecular imaging. On the other hand, nonlinear lightmatter interactions have also proved very useful in microscopy. New harmonic generation microscopy techniques based on focal. Its potential originates from the ability to image in a labelfree manner noncentrosymmetric structures, which exhibit a nonvanishing secondorder susceptibility tensor. We discuss the utility of this technique, as well as the technical issues involved in using this compact source, and demonstrate the first use, to our knowledge, of imaging by thg backlighting.
Highharmonic generation hhg is a coherent frequencyconversion process arising from gaseous atoms 1,2,3,4 or crystalline solids 5,6,7 when they are irradiated by an intense laser pulse. Improving resolution of second harmonic generation microscopy via. 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. Third harmonic thg microscopy nonlinear microscopies have the unique ability to provide micrometerscale 3d images from within complex, scattering samples like biological tissues. Optimization of secondharmonic generation microscopy article pdf available in micron 327. Labelfree second harmonic generation holographic microscopy. In these microscopies, the signal is a coherent superposition of the. Higher harmonic generation microscopy for developmental biology. Third harmonic generation thg microscopy is a labelfree scatter process that is elicited by waterlipid and waterprotein interfaces, including intra and extracellular membranes. Dentin microarchitecture using harmonic generation microscopy. Structure sensitivity in thirdharmonic generation microscopy. 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. Nonlinear optical microscopy nlom is a well established tool for the interrogation of living biological specimen. Optical second harmonic generation microscopy as a tool of.
Chara plant rhizoids have been imaged, showing dynamic plant activity, and nonfading image characteristics even with continuous viewing, indicating prolonged viability under. This feature describes its chemical and physical principles and highlights current applications in disease diagnostics. Second harmonic generation microscopy is a novel technique. Request pdf higher harmonic generation microscopy higher harmonic generation, including second harmonic generation and third harmonic generation, leaves no energy deposition to the interacted. Although the nonlinear optical effect known as secondharmonic generation shg has been recognized since the earliest days of laser physics. In biological tissue, type i and ii fibrillar collagen, microtubule assemblies including centrosomes and mitotic spindles and muscle myosin all endogenously. 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. This unit describes how higher harmonic generation microscopy hhgm is. Pdf secondthird harmonic generation microscopy researchgate. Second harmonic generation imaging distinguishes both high. Second harmonic generation nanoparticles show promise as imaging probes due to their coherent and stable signal with a broad flexibility in the choice of excitation wave. Secondharmonic imaging microscopy shim is based on a nonlinear optical effect known as secondharmonic generation shg. 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.
A secondharmonic microscope obtains contrasts from variations in a specimens ability to generate secondharmonic light from the. The rp photonics buyers guide contains 9 suppliers for high harmonic generation equipment. Second harmonic generation shg microscopy has emerged as a powerful modality for imaging fibrillar collagen in a diverse range of tissues. Third harmonic generation microscopy of cells and tissue organization. Contrary to twophoton excited fluorescence tpef, however, they have found relatively specialized applications, such as imaging collagenous and nonspecific tissues, respectively. There has been increasing interest in second harmonic generation shg imaging approaches for the investigation of atherosclerosis due to the deep penetration and threedimensional sectioning capabilities of the nonlinear optical microscope. Secondharmonic generation imaging of cancer sciencedirect. Graduate institute of electrooptical engineering, national taiwan university, taipei,10617 taiwan, r. 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. Pdf digital holography for second harmonic microscopy. Adaptive optics is implemented in a harmonic generation microscope using a wavefront sensorless correction scheme. Third harmonic generation thg microscopy is a labelfree scatter process that is elicited by. 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. We have recently proposed and demonstrated a novel nonlinear scanning laser microscope that uses third harmonic generation thg to characterize transparent specimen 10.
New harmonic generation microscopy techniques based on focal volume modelling. Spring national institutes of health, bethesda, maryland, u. High orbital angular momentum harmonic generation j. A pictorial representation of the second harmonic generation phenomenon is shown in fig. In addition, results on the recent progress and impact shg microscopy has made in different areas of biology and medicine are presented.
Second harmonic generation microscopy was originally developed alongside of incoherent nonlinear microscopy multiphoton excitation fluorescence, and largely. 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. Higher harmonic generation is a nonlinear coherent scattering process, resulting. The dipoles are oscillated with the applied electric field of frequency w, and it radiates electric field of 2w as well as 1w. Secondharmonic generation microscopy of collagenbearing. By choosing a laser working in the biological penetration window.
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