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

Sang Hoon Choi,Wi-Han Kim,이영중,이호,이원주,양정덕,심종원,김진웅 한국광학회 2011 Current Optics and Photonics Vol.15 No.1

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 µm, 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.

Two-Photon Fluorescent Probes for Biomembrane Imaging: Effect of Chain Length

Kim, Hwan Myung,Kim, Bo Ra,Choo, Hyo-Jung,Ko, Young-Gyu,Jeon, Seung-Joon,Kim, Chul Hoon,Joo, Taiha,Cho, Bong Rae WILEY-VCH Verlag 2008 Chembiochem Vol.9 No.17

<P>Two-photon fluorescent probes for the cellular membrane, derived from 6-acyl-2-aminonaphthalene as the fluorophore and hexanoyl (CH), lauryl (CL), and stearyl (CS) groups as the receptor, have been synthesized. Their photophysical properties and utility as membrane probes were also studied. Whereas CH cannot be used as a membrane probe due to its high water solubility, CL and CS are useful two-photon probes for membrane lateral heterogeneity, as they can easily stain cells, emit fluorescence with high sensitivity to the environment polarity, and are capable of imaging the membrane lateral heterogeneity in live cells. Moreover, CS is more likely to be located in the plasma membrane due to its negligible water solubility. Our results show that the liquid ordered-like domain covers 31–35 % of the cellular surface.</P> <B>Graphic Abstract</B> <P>Two photons are better than one: Two-photon probes have been developed based on 6-acyl-2-aminonaphthalene as the fluorophore and hexanoyl (CH), lauryl (CL), stearyl (CS) groups as membrane receptors. Generalized polarization images of CL- and CS-labeled 293T cells reveal that CS is preferentially located in the plasma membrane, whereas CL is distributed in both plasma membrane and cytosol. Both CL and CS are useful two-photon probes for membrane lateral heterogeneity. <img src='wiley_img/14394227-2008-9-17-CBIC200800353-content.gif' alt='wiley_img/14394227-2008-9-17-CBIC200800353-content'> </P>

Two-Photon Fluorescent Probes for Long-Term Imaging of Calcium Waves in Live Tissue

Kim, Hwan Myung,Kim, Bo Ra,An, Myoung Jin,Hong, Jin Hee,Lee, Kyoung J.,Cho, Bong Rae WILEY-VCH Verlag 2008 Chemistry Vol.14 No.7

<P>2-Acetyl-6-(dimethylamino)naphthalene-derived two-photon fluorescent Ca<SUP>2+</SUP> probes (ACa1–ACa3) are reported. They can be excited by a 780 nm laser beam, show 23–50-fold enhancement in one- and two-photon excited fluorescence in response to Ca<SUP>2+</SUP>, emit fourfold stronger two-photon excited fluorescence than Oregon Green 488 BAPTA-1 upon complexation with Ca<SUP>2+</SUP>, and can selectively detect intracellular free Ca<SUP>2+</SUP> ions in live cells and living tissues with minimum interference from other metal ions and membrane-bound probes. Moreover, these probes are capable of monitoring calcium waves at a depth of 120–170 μm in live tissues for 1100–4000 s using two-photon microscopy with no artifacts of photobleaching.</P> <B>Graphic Abstract</B> <P>Creating a good image: 2-Acetyl-6-(dimethylamino)naphthalene-derived two-photon fluorescent probes for Ca<SUP>2+</SUP> can visualize the calcium waves in live cells and living tissue for a long period of time without mistargeting and photobleaching problems (see picture). <img src='wiley_img/09476539-2008-14-7-CHEM200701453-content.gif' alt='wiley_img/09476539-2008-14-7-CHEM200701453-content'> </P>

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

Yu Na Shin,임창수,Yu Shun Tian,Won Young Rho,조봉래 대한화학회 2010 Bulletin of the Korean Chemical Society Vol.31 No.3

A two-photon fluorescent probe (ACaCL) that can detect near-membrane Ca2+ is reported. This probe can be excited by 780 nm fs pulses, shows high photostability and negligible toxicity, and can visualize near-membrane Ca2+ in live cells and deep inside live tissues by two-photon microscopy.

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 Korean Chemical Society 2010 Bulletin of the Korean Chemical Society Vol.31 No.3

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.

A two-photon ratiometric probe for detection of hNQO1 enzyme activity in human colon tissue

Cho, Myoung Ki,Lim, Chang Su,Sarkar, Avik Ranjan,Lee, Hyo Won,Choi, Hyeon Jin,Noh, Choong-Kyun,Shin, Sung Jae,Kim, Hwan Myung Elsevier 2018 Sensors and actuators. B Chemical Vol.272 No.-

<P><B>Abstract</B></P> <P>Human colonic carcinomas are one of the most common cancers in all Western countries, but there is no reliable method for quantitative and rapid analysis of colon cancer. Herein, we report an hNQO1 enzyme-selective and two-photon emission ratiometric probe (<B>SHC</B>) for quantitative analysis of hNQO1 activity in human colon tissue. This probe shows a blue-to-yellow emission color change in response to hNQO1, high sensitivity and selectivity for hNQO1 over other bio-metabolites, and two-photon microscopy (TPM) imaging capability, thereby allowing precise monitoring of the activity of hNQO1 in situ. Ratiometric TPM imaging with <B>SHC</B> in human colon tissues revealed that hNQO1 activities gradually increased during cancer progression from normal to adenoma, and to adenocarcinoma colon tissues. These findings demonstrated that <B>SHC</B> labeling can detect the differential activities of hNQO1 in the distinct stages of colon neoplasm. Thus, this approach, enzyme-selective TP probe labeling, will likely provide a reliable and fast method for precise diagnosis of colon cancer.</P> <P><B>Highlights</B></P> <P> <UL> <LI> A two-photon emission ratiometric probe for hNQO1 activity was developed. </LI> <LI> The probe shows high sensitivity and selectivity for hNQO1 over other bio-metabolites. </LI> <LI> Quantitative analysis of hNQO1 activity in human colon tissues was demonstrated. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Intravital Laser-scanning Two-photon and Confocal Microscopy for Biomedical Research

Jieun Moon,김필한 대한의학레이저학회 2021 MEDICAL LASERS Vol.10 No.1

Intravital microscopy is a high-resolution imaging technique based on laser-scanning two-photon and confocal microscopy, which allows dynamic 3D cellular-level imaging of various biological processes in a living animal in vivo . This unique capability allows biomedical researchers to directly verify a hypothesis in a natural in vivo microenvironment at the cellular level in a physiological setting. During the last decade, intravital microscopy has become an indispensable technique in several fields of biomedical sciences such as molecular and cell biology, immunology, neuroscience, developmental, and tumor biology. The most distinct advantage of intravital microscopy is its capability to provide a longitudinal view of disease progression at the cellular-level with repeated intravital imaging of a single animal over time by saving the images after each session.

A two-photon ratiometric probe for hydrogen polysulfide (H₂S_n): Increase in mitochondrial H₂S_n production in a Parkinson’s disease model

Choi, Hyeon Jin,Lim, Chang Su,Cho, Myoung Ki,Kang, Ji Su,Park, Soo Jin,Park, Sang Myun,Kim, Hwan Myung Elsevier 2019 Sensors and actuators. B, Chemical Vol.283 No.-

<P><B>Abstract</B></P> <P>Hydrogen polysulfide (H<SUB>2</SUB>S<SUB>n</SUB>, n>1), which is primarily generated during the crosstalk between H<SUB>2</SUB>S and reactive species (ROS and RNS), is receiving increasing attention in biochemical research. H<SUB>2</SUB>S<SUB>n</SUB> is mostly generated in the mitochondria, and abnormal mitochondrial function and oxidative stress are directly related to many disorders including Parkinson's disease (PD). We now report a two-photon fluorescent probe (<B>SPS-M1</B>) for in situ detection of H<SUB>2</SUB>S<SUB>n</SUB> and its application to a PD model to account the H<SUB>2</SUB>S<SUB>n</SUB> levels. The probe exhibited selective and fast response to H<SUB>2</SUB>S<SUB>n</SUB> along with a marked blue-to-green color change. <B>SPS-M1</B> is sensitive enough to quantitative detection of endogenous H<SUB>2</SUB>S<SUB>n</SUB> content in mitochondria using two-photon microscopy (TPM). Ratiometric TPM imaging of live neurons and brain slices using <B>SPS-M1</B> revealed that H<SUB>2</SUB>S<SUB>n</SUB> production is increased to a greater extent in the A53 T α-synuclein (α-syn) overexpressing model than in the wild-type control. These findings suggest that the interactions of H<SUB>2</SUB>S and the increased ROS caused by α-syn overexpression may generate more H<SUB>2</SUB>S<SUB>n</SUB>. By employing our recently published TP probe for mitochondrial H<SUB>2</SUB>S, we also found the relationship between the H<SUB>2</SUB>S<SUB>n</SUB> and H<SUB>2</SUB>S; increased H<SUB>2</SUB>S<SUB>n</SUB> and decreased H<SUB>2</SUB>S levels, indicating that H<SUB>2</SUB>S and H<SUB>2</SUB>S<SUB>n</SUB> may play a significant role in the pathogenesis of PD. This result may be useful to biomedical studies, including PD.</P> <P><B>Highlights</B></P> <P> <UL> <LI> A two-photon excitable and emission ratiometric probe for hydrogen polysulfide (H<SUB>2</SUB>S<SUB>n</SUB>) in mitochondria was developed. </LI> <LI> The probe is able to direct visualization of the endogenous H<SUB>2</SUB>S<SUB>n</SUB> level using two-photon microscopy (TPM). </LI> <LI> Ratiometric TPM imaging revealed that H<SUB>2</SUB>S<SUB>n</SUB> production is increased in a Parkinson’s Disease model. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

이광자 분자센서를 이용한 생체 내 금속이온 검출

김환명(Hwan Myung Kim) 한국청정기술학회 2011 청정기술 Vol.17 No.3

이광자 현미경(two-photon microscopy)은 다양한 생명현상을 살아있는 조직의 깊은 곳에서 관찰할 수 있다는 장점 때문에 차세대 영상기술로 발전하고 있다. 이에 따라 다양한 목적에 사용할 수 있는 이광자 센서의 개발이 활발하게 진행 중이다. 이 총설에서는 금속이온 이광자 센서에 관한 최근의 연구 결과를 소개하고자 한다. 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.

A ratiometric two-photon probe for Ca²⁺ in live tissues and its application to spinal cord injury model

Kim, H.J.,Lim, C.S.,Lee, H.W.,Lee, H.S.,Um, Y.J.,Kumar, H.,Han, I.,Kim, H.M. IPC Science and Technology Press 2017 Biomaterials Vol.141 No.-

Ratiometric imaging with a small-molecule probe is important for the in-situ quantitative analysis of chemical events. We developed a ratiometric two-photon fluorescent probe (SCa1-I<SUB>REF</SUB>) derived from dual dyes with different Stokes shifts. This probe has two identical windows: a Ca<SUP>2+</SUP>-sensing window and an internal reference window, with eliminated FRET interference. SCa1-I<SUB>REF</SUB> shows a marked change in the ratio upon response with Ca<SUP>2+</SUP>, significant two-photon brightness, considerable selectivity for Ca<SUP>2+</SUP>, and cell loading ability with low cytotoxicity. The ratiometric two-photon microscopy images revealed that this probe could directly and quantitatively estimate Ca<SUP>2+</SUP> in live neurons and various tissues including rat spinal cord tissue. The studies of spinal cord injury model revealed that the Ca<SUP>2+</SUP> level was significantly affected by elapsed time after injury. These results will provide useful applications for in-situ [Ca<SUP>2+</SUP>]<SUB>i</SUB> imaging and for the development of effective ratiometric probes.