This study probes the molecular structure of the myofilaments and investigates the movement correlations between sarcomeres during contraction. Resolution and contrast enhancement of subtractive second. The imaging theory of nonlinear second harmonic generation shg and third harmonic generation thg in confocal microscopy is presented in this paper. Secondorder nonlinear holographic microscopy for highspeed, threedimensional imaging is demonstrated. We use photon counting to improve the sn of weak shg signal detection. 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 order of magnitude smaller. The nonlinear imaging theory of second harmonic generation shg and third harmonic generation thg in confocal microscopy is presented in this paper. Higher harmonic generation microscopy for developmental. Labelfree imaging of bone multiscale porosity and interfaces using. In these microscopies, the signal is a coherent superposition of the. Matcher2,3 enhanced image contrast in biological second harmonic imaging microscopy shim has previously. Secondharmonic generation shg microscopy is a label free imaging technique to study collagenous materials in extracellular matrix environment with high resolution and contrast. Add 750 l cellfree collagen stock solution and mix until homogenous slightly.
Osa second harmonic generation microscopy of early. It is shown that this method can resolve interfaces and inhomogeneities with axial resolution comparable to the confocal length of the beam. 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. Among all label free optical tomography techniques, third harmonic generation thg microscopy can achieve better contrast and. The correlated action of hundreds of sarcomeres is needed to produce the myocyte contractions. Secondharmonic generation imaging of membrane potential. Pdf when the energy density at the focal spot of a microscope is. Starch is a major source of our daily diet and it is important to understand the molecular structure that plays a significant role in its wide number of applications. 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. 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. Third harmonic generation microscopy of cells and tissue organization. Second harmonic generation microscopy has emerged as a powerful new optical imaging modality. 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. However, these optical modalities rely on exogenous labeling.
In two photon fluorescence, a fluorophore absorbs two photons at a low. The nonlinear effect of shg and thg on the imaging properties of confocal microscopy has been analyzed in detail by the imaging theory. The extracellular matrix plays an important role in cellular balance, and collagen fibers are its most important component. Labelfree live brain imaging using thirdharmonic generation microscopy. Here we show that third harmonic generation thg microscopy is a suitable tool for label free intravital investigations of the microcirculation in widelyused physiological model systems. Advanced microscopy techniques for revealing molecular. Characterization of oral precancerous lesions based on. Characterization of muscle contraction with second harmonic. Photonics dictionary secondharmonic generation microscopy a nonlinear label free imaging technique commonly used during surgical procedures for the visualization of collagen fibers and muscle tissue myosin with submillimeter resolution. Pdf second harmonic generation microscopy for quantitative.
We have developed this nonlinear optical microscope in order to make possible nondestructive, threedimensional 3d observations of various kinds of inorganic and organic materials. Pdf secondthird harmonic generation microscopy researchgate. Over the last few years, second harmonic generation shg microscopy. Shg transmission images of collagen fibers were spatially resolved due to a coherent, directional shg component. Virtual optical biopsy of human adipocytes with third. We have acquired holograms with acquisition times as short as 10 ms, limited by the switching speed of our shutter.
Second harmonic generation shg can be used for imaging membrane potential in neurons, but poor signaltonoise snlimits accurate measurements of small voltage transients. Biological second and third harmonic generation microscopy. Second harmonic imaging microscopy of living cells pdf. Additivecolor multi harmonic generation microscopy for simultaneous label free differentiation of plaques, tangles, 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. In this study, second harmonic generation shg microscopy is employed for imaging striated myocytes. Pdf optimization of secondharmonic 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. May 01, 2011 second harmonic generation microscopy has emerged as a powerful new optical imaging modality. Shg was first demonstrated by kleinman in crystalline quartz in 1962 kleinman, 1962, and, since that time, has been commonly used to frequency double pulsed lasers to obtain shorter wavelengths. The use of an array of fluorochromes has made it possible to identify cells and submicroscopic cellular components and entities with a high. Pdf higher harmonic generation microscopy of in vitro. Higher harmonic generation microscope hgm, providing images with a higher harmonic generation microscopy hgm can provide a noninvasive biopsy tool with excellent 3d spatial resolution because of its virtual transition and nonlinear characteristics. Optical microscopy davidson and abramowitz optical microscopy. The onehalf wavelength out of phase, caused by the specimen. This work is a major step towards labelfree microscopic live brain imaging, and opens up. Request pdf higher harmonic generation microscopy higher harmonic generation, including second harmonic generation and third harmonic generation, leaves no energy deposition to the interacted.
Most multiphoton microscopy studies in biological systems have relied on twophoton excited fluorescence tpef to produce images. Nonlinear scanning laser microscopy by third harmonic generation. 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. Here, we explore second harmonic generation shg microscopy for visualization of establishing cardiac fibers such as collagen in mouse embryos through the earliest stages of development. Not to be confused with twophoton excitation microscopy. Thg has been applied for labelfree imaging of the brain farrar et al. Because of its underlying physical origin, it is highly sensitive to the collagen fibrilfiber structure, and. Second harmonic generation shg and third harmonic generation thg processes are both nonlinear processes, related to the interaction of intense light with matters.
The use of harmonic generation allows image formation of endogenous features in biological samples such as muscle tissue. Labelfree second harmonic generation holographic microscopy of. Based on digital holography, this technique captures, in singleshot hologram acquisition, both the amplitude and the phase of a coherent shg radiation, which makes possible second harmonic phase microscopy. Using third harmonic generation thg microscopy, we demonstrate that granularity differences of leukocytes can be revealed without a label. In this study, we combined these two approaches by using acellular dermal matrix adm recellularized with mscs to repair cutaneous wounds in a murine model and twophoton fluorescence tpf microscopy and second harmonic generation shg microscopy to assess the effects of this therapy on wound healing. Third harmonic generation near the focal point of a tightly focused beam is used to probe microscopical structures of transparent samples. We have previously developed a new way for nonscanning second harmonic generation shg microscopy opt. 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. Tripling the maximum imaging depth with thirdharmonic. 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. The fibrillar collagen in sinusoids of normal livers could be observed through second harmonic generation shg microscopy. Third harmonic generation microscopy is used to make dynamical images of living systems for the first time.
Studying metalorganic frameworks using second harmonic. Shim has been established as a viable microscope imaging contrast mechanism for visualization of cell and tissue structure and function. Because of its underlying physical origin, it is highly. This feature describes its chemical and physical principles and highlights current applications in disease diagnostics. The cantilever arm has a shape selected to tune the fundamental resonance frequency or a resonance frequency of a selected higher order mode so that the fundamental and higher order resonance frequencies have an integer ratio or near integer ratio. The technique could then be a powerful tool in biomedicine andor in clinical diagnoses. In vivo optical virtual biopsy of human oral mucosa with. Threedimensional highresolution secondharmonic generation. The extracellular matrix ecm, and particularly fibrillar collagen, are central to heart biomechanics, regulating tissue strength, elasticity and contractility. In this work, we present holographic shg phase microscopy. Aug 31, 2016 fully integrated reflectionmode photoacoustic, twophoton and second harmonic generation microscopy in vivo. 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.
Two photon microscopy and second harmonic generation. 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. Optical second harmonic generation microscopy as a tool of. The design and construction of a second harmonic generation. Secondharmonic imaging microscopy shim on a laserscanning system proves.
Previous studies have shown that harmonic generation microscopy hgm, a virtual. Osa labelfree second harmonic generation holographic. A harmonic cantilever for use in an atomic force microscope includes a cantilever arm and a probe tip. Imaging with secondharmonic generation nanoparticles. 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. Second harmonic generation microscopy versus third. Nonlinear microscopy presents numerous advantages over linear microscopy techniques including improved deep tissue. Here, we demonstrate, for the first time, a label free additivecolor multi harmonic generation microscopy to elucidate, concurrently with different hues, alzheimers disease ad neuropathological hallmarks. By adapting a laser scanning microscope with a titanium sapphire femtosecond pulsed laser and transmission optics, we are able to produce live cell images based on the nonlinear optical phenomenon of second harmonic generation shg. Second harmonic generation microscopy versus third harmonic generation microscopy in biological tissues. Second harmonic generation microscopy gets better contrast. Higher harmonic generation microscopy springerlink. A second harmonic microscope obtains contrasts from variations in a specimens ability to generate second harmonic light from the. Imaging granularity of leukocytes with third harmonic.
This unit describes how higher harmonic generation microscopy hhgm is. Second harmonic generation imaging distinguishes both high. Labelfree secondharmonic phase imaging of biological. Thirdharmonic generation microscopy with focusengineered. Fully integrated reflectionmode photoacoustic, twophoton and second harmonic generation microscopy in vivo. Labelfree second harmonic generation holographic microscopy. Pdf in vivo imaging using harmonic generation microscopy. 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 order of. Hematoxylin and eosinstained slides from colon biopsies normal, lowgrade dysplasia lgd, highgrade dysplasia hgd, and. 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. Membrane imaging by simultaneous secondharmonic generation and twophoton microscopy l. Second harmonic imaging microscopy shim is based on a nonlinear optical effect known as second harmonic generation shg. 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.
Determination of blood flow velocity and related hemodynamic parameters is an important aspect of physiological studies which in many settings requires fluorescent labeling. Second harmonic imaging shim is an ideal method for probing membranes of living cells because it offers the high resolution of nonlinear optical microscopy with. The outoffocus image degradation problem of oct also puts an impediment to its application to developmental biology studies. It is thus highly desired to apply hgm for in vivo mucosa histopathological diagnosis. The laser beam was scanned in the x and y axes using a pair of galvanometric mirrors cambridge technologies, inc. Zhilie tang, da xing, and songhao liu the nonlinear imaging theory of second harmonic and third harmonic generations in confocal microscopy. Read simultaneous stimulated raman scattering and higher harmonic generation imaging for liver disease diagnosis without labeling, proceedings of spie on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips. Recent clinical studies on human skin indicated that in vivo multi harmonic generation microscopy hgm can achieve submicron resolution for histopathological analysis with a high penetration depth and leave no energy or photodamages in the interacted tissues.
Second harmonic generation microscopy for quantitative. In prior work, we showed the utility of second harmonic generation imaging. Chara plant rhizoids have been imaged, showing dynamic plant activity, and nonfading image characteristics even with continuous viewing, indicating prolonged viability under. Us7451638b1 harmonic cantilevers and imaging methods for. Secondharmonic generation shg is beginning to emerge as a powerful. Simulation of higher harmonics generation in tappingmode atomic force microscopy article pdf available in applied physics letters 7926. With increasing applications of multiphoton microscopy to thicktissue intravital imaging, second harmonic generation shg from structural proteins has emerged as a potentially important new contrast mechanism. Osa additivecolor multiharmonic generation microscopy.
It is proved that the imaging process of shg and thg in confocal microscopy, which is different from. Secondharmonic generation microscopy for assessment of. Nonlinear optical microscopy by multiphoton excitation has. 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. 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.
We have recently proposed and demonstrated a novel nonlinear scanning laser microscope that uses third harmonic generation thg to characterize transparent specimen 10. Second harmonic generation microscopy is a label free optical imaging technology which can detect and quantitate collagen in tissue sections without immunostaining. In this study, we combined these two approaches by using acellular dermal matrix adm recellularized with mscs to repair cutaneous wounds in a murine model and twophoton fluorescence tpf microscopy and second harmonic generation shg microscopy. In biological tissue, type i and ii fibrillar collagen, microtubule assemblies including centrosomes and mitotic spindles and muscle myosin all endogenously. We performed second harmonic generation shg imaging of collagen in rattendon cryosections, using femtosecond laser scanning confocal microscopy, both in backscattering and transmission geometries. To satisfy the nyquist criterion for both tpm and thg, a pixel size of 0.
Sheppard, second harmonic imaging in the scanning optical microscope, opt. Higher harmonic generation microscopy for developmental biology. 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. Least invasive in vivo imaging using harmonic generation. Higher harmonic generation is a nonlinear coherent scattering process. Second harmonic generation shg microscopy has emerged as a powerful modality for imaging fibrillar collagen in a diverse range of tissues. All these imaging modalities are compatible with twophoton excited. With these advanced microscopy techniques 1824, we can characterize the 3d morphology of adipocytes without labeling.
Estimation of helical angles of myosin and collagen by second. Second harmonic generation confocal microscopy of collagen. Commonly used methods, histological staining followed by either semiquantitative or computerized image analysis, have limited sensitivity, accuracy, and operatordependent variations. Keywords nonlinear microscopy techniques, second harmonic generation shg imaging microscopy, multiphoton microscopy, breast cancer, ovarian cancer introduction ovarian and breast cancers account for 31% of all cancerassociated deaths each year despite the respective high 5year survival rates of 92% and 99% when diagnosed in early stages. Optical microscopy davidson and abramowitz 3 degrees out of step more commonly, out of phase with the direct light that has passed through undeviated. A new mode of contrast in biological second harmonic. Imaging cells and extracellular matrix in vivo by using. Enhancing resolution and contrast in secondharmonic. Estimation of helical angles of myosin and collagen by second harmonic generation imaging microscopy franc. A study on image quality in polarizationresolved second harmonic generation microscopy. Article views are the countercompliant sum of full text article downloads since november 2008 both pdf and html across all institutions and individuals. In vivo imaging using harmonic generation microscopy. This effect was enhanced with the use of an indexmatching fluid ni1. Simultaneous stimulated raman scattering and higher.
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. Pdf simulation of higher harmonics generation in tapping. Labelfree determination of hemodynamic parameters in the. Fully integrated reflectionmode photoacoustic, twophoton. Higher harmonic generation microscopy of in vitro cultured mammal oocytes and embryos. Label free imaging techniques, such as coherent antistokes raman scattering microscopy and second harmonic generation microscopy can. Second harmonic generation shg is beginning to emerge as a powerful contrast mechanism in nonlinear optical microscopy. Raman scattering cars, secondharmonic generation shg or thirdharmonic generation thg are receiving considerable attention. New harmonic generation microscopy techniques based on focal. Multiphoton microscopy and second harmonic generation loci. 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 microscopy is a novel technique. Shg has the unique ability to evaluate the amount of extracellular matrix collagen protein and its alignment. Structure sensitivity in thirdharmonic generation microscopy. Membrane imaging by simultaneous secondharmonic generation. Imaging of biological membranes in liquid institute for biophysics johannes kepler university of linz altenbergerstr. The nonlinear imaging theory of second harmonic and third. A new mode of contrast in biological second harmonic generation microscopy. Laser scanning thirdharmonicgeneration microscopy in biology. We extend the subtractive imaging method to label free second harmonic generation shg microscopy to enhance the spatial resolution and. Second harmonic generation microscopy reveals altered.
Higher collagen signals are observed in breast fibroadenomas in comparison to. Highresolution nonlinear optical imaging of live cells by. Labelfree live brain imaging and targeted patching with third. Secondharmonic imaging microscopy shim is based on a nonlinear optical effect known as secondharmonic generation shg. Jan 14, 2014 the framework is the first to include reflection and refraction of the excitation beam and nonlinear signals by an arbitrary number of interfaces in the focal volume, which is especially significant for the interpretation of third harmonic generation thg. The quadratic intensity dependence of shg is exploited to preferentially reveal unscattered signal light and reject outoffocus scattered background.
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