Evaluation of newly synthesized potential NLO-phores for 2-photon and SHG imaging
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Our contribution is focused on broadening of the spectrum of available non-linear optical (NLO)-phores (contrast agents for nonlinear optical microscopy) by design and synthesis of new organic dyes with appropriate optical properties. One of the main pre-requisites of microscopy utilizing non-linear excitation is the existence of molecules that are able to provide NLO response for the second-harmonic generation (SHG) or for the two-photon excited fluorescence (TPEF). Many molecules naturally occurring in living tissue such as collagens or NAD(P)H were successfully used in this regard, but there is a natural interest in broadening of the spectrum of available NLO-phores. Gathered results confirm applicability of the newly synthesized dyes as new potential NLO-phores for confocal laser scanning microscopy with nonlinear excitation in rat aorta.Keywords:
Two-photon excitation microscopy
Second-harmonic imaging microscopy
Theoretical study and numerical simulation were carried out on the newly proposed way of cascaded second harmonic generation (SHG) to get stable SHG output. The results certify that by way of using cascaded SHG one can obtain stable SHG output. Our results also show that by tuning the angle between k and the optical axis and the distance between the two SHG crystals, the length of the second SHG crystal for most stable SHG can also be tuned. When the length for most stable SHG is tuned to the real length of the second SHG crystal, stable SHG output was be obtained. Both stable SHG output and high SHG conversion efficiency can be got using this new way, and this will help a lot to design the pumping system for the optical pulse chirped amplifying system.
Second-harmonic imaging microscopy
Crystal (programming language)
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Second-harmonic imaging microscopy
Supercontinuum
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Photobleaching
Two-photon excitation microscopy
Phototoxicity
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As a new technology of nonlinear microscopic imaging,second harmonic generation(SHG) have been widely used in all fields of medicine and biology.In this paper,we analyzed the principle of SHG,introduced the setup and development of SHG technology.Then we made a comparison between SHG and two-photon excited fluorescence(TPEF).Some applications in biomedicine were also given.Lastly we predicted the further development and application of SHG.
Second-harmonic imaging microscopy
Two-photon excitation microscopy
Biomedicine
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We report the use of Interferometric Second Harmonic Generation (I-SHG) microscopy to eliminate imaging artifacts from SHG patterns. Boundaries between two regions of opposite polarities in periodically-poled lithium niobate (PPLN) and in muscle myosin are compared, and shown to produce respectively incoherent contributions to the SHG signal and artefactual interferences. I-SHG allows to remove those artifacts, which opens a great opportunity for a better characterization of SHG images of various materials, especially complex media like biological tissues.
Second-harmonic imaging microscopy
Characterization
SIGNAL (programming language)
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Second harmonic generation(SHG) imaging has the advantages of large penetration depth,high spatial and temporal resolution and low photodamage.Because of its sensitivity to the symmetry change of the microstructure,SHG imaging technique becomes to be one of the hot topics in bio-imaging research.The principle and the applications of SHG imaging are demonstrated in this paper,and the focus of SHG technique is on the development of intracellular SHG imaging that various labels,including voltage sensitive dyes,chiral molecule and metal nanoparticles are used.
Second-harmonic imaging microscopy
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Abstract In the past several years, second-harmonic generation (SHG) has emerged as a powerful nonlinear optical contrast mechanism for biological imaging applications. SHG is a coherent processwhere two lower-energy photonsare up-converted to exactly twice the incident frequency (or half the wavelength). This effect was first demonstrated by Kleinman (Kleinman, 1962) in crystalline quartz in 1962, where thisadvance wasmade possible with the invention of the ruby laser. Since that discovery, SHG in uni-axial birefringent crystals has been exploited to frequency double pulsed lasers to obtain shorter wavelengths, thereby producing multiple colors from a single source. SHG from interfaces was later discovered by Bloembergen in 1968 and rapidly became a versatile spectroscopic tool to study chemical and physical processes at air–solid, air– liquid, and liquid–liquid interfaces (for reviews, see Eisenthal, 1996; Shen, 1989). The first integration of SHG and optical microscopy was achieved in 1974 by Hellwarth and coworkers, who used SHG as an imaging tool to visualize the microscopic crystal structure in polycrystalline ZnSe (Hellwarth & Christensen, 1974). Sheppard then implemented the method on a scanning microscope in 1977 (Sheppard et al., 1977).
Second-harmonic imaging microscopy
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Chiral molecules have different efficiencies in generating second-harmonic generation signal for left- and right-circular-polarized light. This effect is called second-harmonic generation circular dichroism (SHG-CD). It has been shown that SHG-CD exhibits much better chiral contrast than traditional chiroptical spectroscopies [1]. Furthermore, combined with a laser scanning microscope, SHG-CD provides optical sectioning capability that is suitable for examining thick tissue samples. We have shown that type I collagen gives rise to strong second-harmonic generation circular dichroism (SHG-CD) responses [2]. However, to resolve the molecular structures and chiral properties of biological tissues, it is not enough to study SHG-CD for only one specific wavelength.
Second-harmonic imaging microscopy
Harmonic
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Potassium titanyl phosphate (KTiOPO4, KTP) particle of nanometric size (nano-KTP) is an attractive material for nonlinear microscopy, and the optimized growth of large-size KTP single crystals has numerous applications for efficient frequency conversion in laser technology. Its three-dimensional orientation and nanoscale morphology are important for growth optimization. In this paper, we introduce an imaging technique based on circular dichroism second-harmonic generation (CD-SHG) to characterize the 3D distribution of KTP nanocrystal. A rigorous theoretical model of CD-SHG imaging for nano-KTP through stratified media is demonstrated. Circular dichroism analysis is used to probe the orientation of 3-axis with respect to the optical observation axis. The research results show that the azimuthal angle of the peak value (SHG) or valley value (CD-SHG) is strongly related to the excitation polarization when the KTP sample is excited by different circular polarizations. Importantly, the refractive index mismatches and the imaging depth also affect the azimuthal angle. Thus, the proposed framework enables a more precise quantitative analysis of the CD-SHG signal of KTP.
Second-harmonic imaging microscopy
Potassium titanyl phosphate
Linear dichroism
Dichroism
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Nonlinear optical imaging has revolutionized microscopy for the life sciences. Second harmonic generation (SHG), the younger sibling of two-photon excited fluorescence (2PF), is a technique that can produce high resolution images from deep inside biological tissues. Second harmonic light is generated by the coherent scattering of an ensemble of aligned chromophores in a focused, pulsed laser beam. SHG is only generated at the focal spot, reducing the background signal, and requires ordered chromophores, so is highly structure-specific. In contrast to two-photon fluorescence, the physical process that creates the signal does not require the formation of excited states, allowing elimination of harmful photochemistry. While the SHG of native proteins and biopolymers is well known, the use of exogenous dyes can provide SHG contrast from areas without a sufficiently high intrinsic quadratic hyperpolarizability, β. Dyes for SHG primarily target lipid bilayers; a trait that, combined with sensitivity to transmembrane potential, allows monitoring of action potentials in a variety of excitable cells, most importantly mammalian neurons. This article summarizes the principles of SHG imaging and explores approaches for maximizing the SHG signal from a biological specimen. We survey methods of optimizing the optical set-up, enhancing the β of the dye and achieving biological compatibility. In conclusion, we examine novel applications of SHG imaging and highlight promising directions for the development of the field.
Second-harmonic imaging microscopy
Hyperpolarizability
Biological Imaging
Two-photon excitation microscopy
Chromophore
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