Both the second and third harmonic intensity signals are used as the optimization metric. Image stitched in fiji from zstacks of 20 region of interests. Here we demonstrate highcontrast imaging of live brain tissue at cellular. To generate high harmonics like n 30 and so on, a substantially different. The extracellular matrix plays an important role in cellular balance, and collagen fibers are its most important component. Shim has been established as a viable microscope imaging contrast mechanism for visualization of cell and tissue structure and function. Biological second and third harmonic generation microscopy. It includes many nonlinear contrast mechanisms, each sensitive to different biological structures.
In prior work, we showed the utility of second harmonic generation imaging microscopy shim in. In prior work, we showed the utility of secondharmonic generation imaging microscopy shim in. New harmonic generation microscopy techniques based on. While this visible transformation is welldescribed, the underlying and causative microscopic changes, in which collagen plays a major role, are poorly understood and difficult to visualise.
Second harmonic generation microscopy was originally developed alongside of incoherent nonlinear microscopy multiphoton excitation fluorescence, and largely. Characterization of photodamage in coherent antistokes. Higher harmonic generation microscopy for developmental. Secondharmonic generation shg, also called frequency doubling is a nonlinear optical process in which two photons with the same frequency interact with a nonlinear material, are combined, and generate a new photon with twice the energy of the initial photons equivalently, twice the frequency and half the wavelength, that conserves the coherence of the excitation. Secondharmonic imaging microscopy shim is based on a nonlinear optical effect known as secondharmonic generation shg. Pdf secondthird harmonic generation microscopy researchgate. Unlocking higher harmonics in atomic force microscopy with. Timevarying tipsample force measurements and steady. Secondharmonic generation is a nonlinear optical process in which two photons with the same. In second harmonic generation shg, laser light is focused on a sample to generate frequencydoubled light.
Continuous wave sum frequency generation and imaging of. 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. Second harmonic imaging microscopy of living cells pdf. Optical microscopy davidson and abramowitz optical microscopy. In vivo imaging of myelin in the vertebrate central nervous. Direct timevarying tipsample force measurements by torsional harmonic cantilevers facilitate detailed investigations of the cantilever dynamics in tappingmode atomic force microscopy.
Tpef 1, 2, second harmonic generation shg 3, 4, and coherent antistokes raman scattering cars microscopy 5, have opened a new window for 3d imaging of biological samples. Pselike zones were presenting shg signal with a higher single band frequency than the control muscles 87. Here, we report experimental evidence that the mathematical relationships describing the steadystate dynamics are quantitatively. Harmonic generation is attractive for in vivo imaging. High contrast 3d imaging of surfaces near the wavelength limit using tabletop euv ptychography. Second harmonic imaging microscopy shim involves illuminating noncentrosymmetric structures with an ultrashort pulse of focused laser. Timevarying tipsample force measurements and steadystate. Electronic microscopy was applied on the same samples to improve shg images interpretation. 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. Graduate institute of electrooptical engineering, national taiwan university, taipei,10617 taiwan, r. In contrast to fluorescence imaging, shg specifically visualizes the plasma membrane at locations that are not attached to artificial substrates and allows. Second harmonic imaging microscopy shim is based on a nonlinear optical effect known as second harmonic generation shg. Kgaa,weinheim 1243 timeresolved second harmonic generation nearfield scanning optical microscopy richardd.
In contrast, thirdharmonic generation thg is a nonlinear optical process ubiquitous to all molecules in which three photons at the fundamental frequency are upconverted to a photon at the third harmonic with an efficiency proportional to the thirdorder susceptibility tensor, x 3. Unlocking higher harmonics in atomic force microscopy with gentle interactions sergiosantos,1, victorbarcons1, josepfont1 andalbertverdaguer2,3 full research paper open access address. Here, we propose to externally drive an arbitrary number of exact higher harmonics above the noise level. Analysis of secondharmonicgeneration microscopy in a. Over the last few years, second harmonic generation shg microscopy has been used for the analysis of collagen fibers in several types of gynaecological cancers, such as breast and ovarian cancer. Polarizationresolved secondharmonicgeneration imaging. Eisenthal,y and rafael yuste howard hughes medical institute, department of biological sciences, andydepartment of chemistry, columbia university, new york. Second harmonic generation microscopy was originally developed alongside of incoherent nonlinear microscopy multiphoton excitation. Live tissue intrinsic emission microscopy using multiphotonexcited native fluorescence and second harmonic generation warren r. Analysis of second harmonic generation microscopy in a mouse model of ovarian carcinoma jennifer m. The unique configuration of the integrated microscope allows for the acquisition of both anatomical and functional imaging information with particular emphasis in the fields of tissue engineering and tumor biology.
Thirdharmonic generation microscopy with focusengineered. High contrast 3d imaging of surfaces near the wavelength limit using tabletop euv ptychography bosheng zhangn, dennis f. Threedimensional highresolution secondharmonic 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. The outoffocus image degradation problem of oct also puts an impediment to its application to developmental biology studies. Timevarying tipsample force measurements and steadystate dynamics in tappingmode atomic force microscopy ozgur sahin the rowland institute at harvard, harvard university, cambridge, massachusetts 02142, usa. Realtime secondharmonicgeneration microscopy based on.
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. Highresolution plasma membraneselective imaging by second. Pdf study of apoptosis induction using fluorescent and. Aberration correction is performed to compensate both system and specimeninduced aberrations by using an efficient optimization routine based upon zernike polynomial modes. By choosing a laser working in the biological penetration window.
Realtime secondharmonicgeneration microscopy based on a 2ghz repetition rate ti. Over the last few years, second harmonic generation shg microscopy has. Second harmonic generation microscopy in zebrafish. Secondharmonic imaging microscopy shim is based on a nonlinear optical effect known as. Higher harmonic generation microscopy hhg 1, which combines the second and third harmonic generation modalities based on 12301250 nm, and can provide high penetration, high resolution and greeshma rajan et al, international journal of computer science and mobile computing, vol. Request pdf higher harmonic generation microscopy higher harmonic generation, including second harmonic generation and third harmonic generation, leaves no energy deposition to the interacted.
Nonlinear microscopy has become an indispensable tool in the study of biological systems. The nonlinear harmonic generation is a function of the molecular structure. Second harmonic generation microscopy for quantitative. Laser scanning thirdharmonicgeneration microscopy in biology. Enhanced higherharmonic imaging in tappingmode atomic force. Electrooptic mechanism of membrane potential sensitivity jiang jiang, kenneth b. Second harmonic generation microscopy of the larval zebrafish tail. Adams jila, university of colorado, 440 ucb, boulder, co 803090440, usa article info article history. Polarizationresolved second harmonic microscopy in collagenous. 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. We have recently proposed and demonstrated a novel nonlinear scanning laser microscope that uses thirdharmonic generation thg to characterize transparent specimen 10. Recent studies in mice and humans have shown that second harmonic generation. This method was found to be particularly useful for volumetric mouse brain imaging 9,10, because outoffocus background generation can be further reduced due to the higher order nonlinear excitation.
Thirdharmonic generation thg microscopy can be complementary to shg. All materials possess thirdorder nonlinear susceptibility and higherorder terms. Enhanced higherharmonic imaging in tappingmode atomic. Highresolution plasma membraneselective imaging by.
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 microscopy davidson and abramowitz optical. Williams, richard christie, alexander yu nikitin, bradley t. However, the technical challenges associated with this modality have limited its broad adoption as a tool for in vivo studies. Oct 21, 2016 imaging of collagen with second harmonic generation microscopy. Imaging of biological membranes in liquid institute for biophysics johannes kepler university of linz altenbergerstr. We systematically investigate higher harmonics in the vibration spectrum of scanning force microscope cantilevers operating in the attractive regime. Polarizationsensitive sumfrequency generation microscopy. The former is a manual method only valid for a defined set of cover thickness values.
Pdf optimization of secondharmonic generation microscopy. Analysis of secondharmonicgeneration microscopy in a mouse. Secondharmonic generation shg microscopy is a powerful microscopy technique for. What links here related changes upload file special pages permanent link. Barton auniversity of arizona, biomedical engineering, 1657 e. Several laserscanning microscopy techniques have been developed in the last twenty years based on nonlinear optical phenomena. Tripling the maximum imaging depth with thirdharmonic 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. 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. 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. Here we demonstrate the first photomultiplierbased realtime second harmonic generation microscopy taking advantage of the strongly. Here we demonstrate the first photomultiplierbased realtime secondharmonicgeneration microscopy taking advantage of. 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. Twophoton excited fluorescence microscopy in conventional confocal laser scanning microscopy, the fluorescence excitation source is a tightly focused continuouswave cw laser beam. Osa polarized secondharmonic generation optical microscopy.
B three views of 3d reconstruction of collagen fibers in an unwounded zebrafish larval tail 3 dpf to illustrate organization of fibers. 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 disorders. Mar 27, 2020 the extracellular matrix plays an important role in cellular balance, and collagen fibers are its most important component. In this kind of atomic force microscopy, if the probes higherorder resonance frequencies match the integer multiples of its fundamental frequency, the probes responses at such harmonic. Higher harmonic generation microscopy springerlink. Kgaa,weinheim 1243 timeresolved second harmonic generation nearfield scanning.
Cars is a fourwave mixing process in which the interaction of a pump. 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. Tripling the maximum imaging depth with thirdharmonic. Pdf when the energy density at the focal spot of a microscope is sufficiently. 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. Second harmonic generation shg microscopy has emerged as a powerful modality for imaging fibrillar collagen in a diverse range of tissues. The onehalf wavelength out of phase, caused by the specimen. Both the second and thirdharmonic intensity signals are used as the optimization metric. In vivo imaging of myelin in the vertebrate central nervous system using third harmonic generation microscopy matthew j. However, interpretation of the images generated in nonlinear microscopy is a complex matter due to factors such as the structural complexity of the. Labelfree live brain imaging and targeted patching with thirdharmonic. Realtime secondharmonicgeneration microscopy based on a. During labour, the cervix undergoes a series of changes to allow the passage of the fetoplacental unit.
Fluorescence microscopy has become an essential tool in biology as well as in materials science as it has attributes that are not readily available in other optical microscopy techniques. Secondharmonic generation shg microscopy has emerged as a powerful modality for imaging fibrillar collagen in a diverse range of tissues. Because of its underlying physical origin, it is highly sensitive to the collagen fibrilfiber structure, and. In dynamic atomic force microscopy, nanoscale properties are encoded in the higher harmonics. The use of an array of fluorochromes has made it possible to identify cells and submicroscopic cellular components and entities with a high. Aug 31, 2016 fully integrated reflectionmode photoacoustic, twophoton and second harmonic generation microscopy in vivo skip to main content thank you for visiting. Second harmonic generation microscopy has been applied on pork semimembranosus samples fixed at 24 hours post mortem. Study of apoptosis induction using fluorescent and higher harmonic generation microscopy techniques in acartia tonsa nauplii exposed to chronic concentrations of nickel. We have recently proposed and demonstrated a novel nonlinear scanning laser microscope that uses third harmonic generation thg to characterize transparent specimen 10. Labelfree live brain imaging using thirdharmonic generation microscopy. Secondharmonic generation shg microscopy is a powerful microscopy technique for imaging collagen and other biological molecules. The high intensity achievable with focussed lasers makes. We show that bright, highresolution 3d shg images of structural proteins within connective tissue.
A new mode of contrast in biological second harmonic generation. Recently, we proposed a method in which an ordinary cantilever is excited at or close to a submultiple of its resonant frequency to enhance any one of the higher harmonics. We show that bright, high resolution 3d shg images of structural proteins within connective tissue. Holographic second harmonic generation microscopy infoscience. Fully integrated reflectionmode photoacoustic, twophoton and second harmonic generation microscopy in vivo skip to main content thank you for visiting. Two photon microscopy and second harmonic generation. Raman scattering cars, secondharmonic generation shg or thirdharmonic generation thg are receiving considerable attention. For nonlinear microscopy, a specimen with a nonlinear optical crosssection will produce higher harmonic light emission under suf.
Pointscanning sumfrequency generation sfg microscopy enables the generation of images of collagen i fibers in tissues by tuning into specific vibrational resonances of the polypeptide. Timeresolved second harmonic generation nearfield scanning. 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. Analysis of secondharmonicgeneration microscopy in a mouse model of ovarian carcinoma jennifer m. Adaptive harmonic generation microscopy of mammalian embryos. Second harmonic generation microscopy gets better contrast. Harmonic generation is a nonlinear optical process in which n photons with the same. Second harmonic generation microscopy reveals collagen. All these imaging modalities are compatible with twophoton excited. Optical microscopy davidson and abramowitz 3 degrees out of step more commonly, out of phase with the direct light that has passed through undeviated. Among these tools, cars microscopy permits molecular imaging without any labeling 616.
This unit describes how higher harmonic generation microscopy hhgm is applied to detect native. In two photon fluorescence, a fluorophore absorbs two photons at a low. Higher harmonic generation microscopy for developmental biology article in journal of structural biology 1471. Nevertheless, when gentle interactions and minimal invasiveness are required, these harmonics are typically undetectable. Realtime secondharmonicgeneration microscopy based on a 2. Adaptive optics is implemented in a harmonic generation microscope using a wavefront sensorless correction scheme. Second harmonic generation shg, also called frequency doubling is a nonlinear optical process in which two photons with the same frequency interact with a nonlinear material, are combined, and generate a new photon with twice the energy of the initial photons equivalently, twice the frequency and half the wavelength, that conserves the coherence of the excitation. Labelfree live brain imaging and targeted patching with third. Fully integrated reflectionmode photoacoustic, twophoton. A secondharmonic microscope obtains contrasts from variations in a specimens ability to generate. Polarized secondharmonic 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.
In other cases, like secondharmonic imaging microscopy, only a tiny fraction of the light. A diagram of zebrafish larvae showing region imaged. A remarkable corollary of this is that a much larger group of peptides and. High contrast 3d imaging of surfaces near the wavelength. We identify and explore a high orbital angular momentum oam harmonics generation and amplification mechanism that manipulates the oam independently of any other laser property, by preserving the initial laser wavelength, through stimulated raman backscattering in a plasma. Probing attractive forces at the nanoscale using higher. It is shown that when collagenrich tissues are visualized near the 2954 cm1 stretching vibration of methylene groups, the sfg image contrast is higher compared to the. Second harmonic generation microscopy reveals collagen fibres. Greens function formulation for thirdharmonic generation microscopy. Polarized secondharmonic generation optical microscopy. In vivo imaging of myelin in the vertebrate central.
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