• Title, Summary, Keyword: Two-Photon Microscopy

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Visualization of Epidermis and Dermal Cells in ex vivo Human Skin Using the Confocal and Two-photon Microscopy

  • Choi, Sang-Hoon;Kim, Wi-Han;Lee, Yong-Joong;Lee, Ho;Lee, Weon-Ju;Yang, Jung-Dug;Shim, Jong-Won;Kim, Jin-Woong
    • Journal of the Optical Society of Korea
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    • v.15 no.1
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    • pp.61-67
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    • 2011
  • The confocal laser scanning microscopy and two-photon microscopy was implemented based on a single laser source and an objective lens. We imaged and compared the morphology of identical sites of ex vivo human skin using both microscopes. The back-scattering emission from the sample provided the contrast for the confocal microscopy. The intrinsic autofluorescence and the second harmonic generation were used as the luminescence source for the two-photon microscopy. The wavelength of the Ti:Sapphire laser was tuned at 710 nm, which corresponds to the excitation peak of NADH and FAD in skin tissue. The various cell layers in the epidermis and the papillary dermis were clearly distinguished by both imaging modalities. The two-photon microscopy more clearly visualized the intercellular region and the nucleus of the cell compared to the confocal microscopy. The fibrous structures in the dermis were more clearly resolved by the confocal microscopy. Numerous cells in papillary dermal layer, as deep as $100\;{\mu}m$, were observed in both CLSM and two-photon microscopy. While most previous studies focused on fibrous structure imaging (collagen and elastin fiber) in the dermis, we demonstrated that the combined imaging with the CLSM and two-photon microscopy can be applied for the non-invasive study of the population, distribution and metabolism of papillary dermal cells in skin.

Detection of Intracellular Free Metal Ions with Molecular Two-Photon Sensors (이광자 분자센서를 이용한 생체 내 금속이온 검출)

  • Kim, Hwan-Myung
    • Clean Technology
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    • v.17 no.3
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    • pp.231-237
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    • 2011
  • Two-photon microscopy (TPM) is attracting much attention in biological imaging due to the capability of imaging deep inside the living tissues for a long period of time. For maximum utilization of TPM, it is essential to develop efficient twophoton sensors. Regarding this, many research groups are developing two-photon sensors for specific applications. In this review, we summarize recent results on selected examples of two-photon sensors for intracellular free metal ions in the live cells and tissues to provide a guideline for various imaging applications.

Detection of Near-membrane Calcium Ions in Live Tissues with a Two-Photon Fluorescent Probe

  • Shin, Yu-Na;Lim, Chang-Su;Tian, Yu Shun;Rho, Won-Young;Cho, Bong-Rae
    • Bulletin of the Korean Chemical Society
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    • v.31 no.3
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    • pp.599-605
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    • 2010
  • A two-photon fluorescent probe (ACaCL) that can detect near-membrane $Ca^{2+}$ is reported. This probe can be excited by 780 nm fs pulses, shows high photostability and negligible toxicity, and can visualize near-membrane $Ca^{2+}$ in live cells and deep inside live tissues by two-photon microscopy.

High-speed Two-photon Laser Scanning Microscopy Imaging of in vivo Blood Cells in Rapid Circulation at Velocities of Up to 1.2 Millimeters per Second

  • Boutilier, Richard M.;Park, Jae Sung;Lee, Ho
    • Current Optics and Photonics
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    • v.2 no.6
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    • pp.595-605
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    • 2018
  • The two-photon process of microscopy provides good spatial resolution and optical sectioning ability when observing quasi-static endogenous fluorescent tissue within an in vivo animal model skin. In order to extend the use of such systems, we developed a two-photon laser scanning microscopy system capable of also capturing $512{\times}512$ pixel images at 90 frames per second. This was made possible by incorporating a 72 facet polygon mirror which was mounted on a 55 kRPM motor to enhance the fast-scan axis speed in the horizontal direction. Using the enhanced temporal resolution of our high-speed two-photon laser scanning microscope, we show that rapid processes, such as fluorescently labeled erythrocytes moving in mouse blood flow at up to 1.2 mm/s, can be achieved.

Two-photon probes for biomedical applications

  • Lim, Chang Su;Cho, Bong Rae
    • BMB Reports
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    • v.46 no.4
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    • pp.188-194
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    • 2013
  • Two-photon microscopy (TPM), which uses two photons of lower energy as the excitation source, is a vital tool in biology and clinical science, due to its capacity to image deep inside intact tissues for a long period of time. To make TPM a more versatile tool in biomedical research, we have developed a variety of two-photon probes for specific applications. In this mini review, we will briefly discuss two-photon probes for lipid rafts, lysosomes, mitochondria, and pH, and their biomedical applications.

Variations of imaging depth and chloroplast emission spectrum of Arabidopsis thaliana with excitation wavelength in two-photon microscopy (이광자현미경 여기 광 파장에 따른 Arabidopsis thaliana 촬영 깊이 및 엽록체 형광 스펙트럼의 변화)

  • Joo, Yongjoon;Son, Si Hyung;Kim, Ki Hean
    • Journal of the Korean Society of Visualization
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    • v.12 no.3
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    • pp.9-14
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    • 2014
  • Two-photon microscopy (TPM) has been used in plant research as a high-resolution high-depth 3D imaging modality. However, TPM is known to induce photo-damage to the plant in case of long time exposure, and optimal excitation wavelength for plant imaging has not been investigated. Longer excitation wavelength may be appropriate for in vivo two-photon imaging of Arabidopsis thaliana leaves, and effects of longer excitation wavelength were investigated in terms of imaging depth, emission spectrum. Changes of emission spectrum as a function of exposure time at longer excitation wavelength were measured for in vivo longitudinal imaging. Imaging depth was not changed much probably because photon scattering at the cell wall was a limiting factor. Chloroplast emission spectrum showed its intensity peak shift by 20 nm with transition of excitation wavelength from 849 nm or below to 850 nm or higher. Emission spectrum showed different change patterns with excitation wavelengths in longitudinal imaging. Longer excitation wavelengths appeared to interact with chloroplasts differently in comparison with 780 nm excitation wavelength, and may be good for in vivo imaging.

Polarity Probing Two-Photon Fluorophores Based on [2.2]Paracyclophane

  • Woo, Han-Young;Korystov, Dmitry;Jin, Young-Eup;Suh, Hong-Suk
    • Bulletin of the Korean Chemical Society
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    • v.28 no.12
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    • pp.2253-2260
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    • 2007
  • A series of tetra donor substituted [2.2]paracyclophane-based two-photon absorption (TPA) fluorophores were synthesized in neutral and cationic forms. The imaging activity of overall set of fluorophores was studied by the two-photon induced fluorescence (TPIF) method in a range of solvents. We also measured a clear progression toward a longer photoluminescence lifetime with increasing solvent polarity (intrinsic photoluminescence lifetime, τi: ~2 ns in toluene → 12-16 ns in water). The paracyclophane fluorophores with this unique property can be utilized as an optical polarity probe for the biomolecular substrates. The combined measurement of the two-photon fluorescence microscopy (TPM) cell image and TPIF lifetime can give us a better understanding of the biological processes and local environments in the cells.

Development of line-scanning two-photon microscopy based on spatial and temporal focusing for tryptophan based auto fluorescence imaging (고속 트립토판 자가형광 이미징을 위한 시공간적 집중 기반의 라인 스캐닝 이광자 현미경 개발)

  • Lee, Jun Ho;Nam, Hyo Seok;Kim, Ki Hean
    • Journal of the Korean Society of Visualization
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    • v.11 no.2
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    • pp.41-45
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    • 2013
  • Two-photon microscopy (TPM) is minimally-invasive 3D fluorescence microscopy based on nonlinear excitation, and TPM can visualize cellular structures based on auto-fluorescence. Line-scanning TPM is one of high-speed TPM methods without sacrificing the image resolution by using spatial and temporal focusing. In this paper, we developed line-scanning TPM based on spatial and temporal focusing for auto-fluorescence imaging by exciting the tryptophan. Laser source for this system was an optical parametric oscillator (OPO) and it made near 570 nm femtosecond pulse laser. It had 200fs pulse width and 1.72 nm bandwidth, so that the achievable depth resolution was 2.41um and field of view (FOV) is 10.8um. From the characterization, our system has 3.0 um depth resolution and 12.3 um FOV. We visualized fixed leukocyte cell sample and compared with point scanning system.