• Molecules and Cells
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• v.26 no.2
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• pp.113-120
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• 2008

Laser light microscopy enables observation of various simultaneously occurring events in living cells. This capability is important for monitoring the spatiotemporal patterns of the molecular interactions underlying such events. Two-photon excited fluorescence microscopy (two-photon microscopy), a technology based on multiphoton excitation, is one of the most promising candidates for such imaging. The advantages of two-photon microscopy have spurred wider adoption of the method, especially in neurological studies. Multicolor excitation capability, one advantage of two-photon microscopy, has enabled the quantification of spatiotemporal patterns of $[Ca^{2+}]_i$ and single episodes of fusion pore openings during exocytosis. In pancreatic acinar cells, we have successfully demonstrated the existence of "sequential compound exocytosis" for the first time, a process which has subsequently been identified in a wide variety of secretory cells including exocrine, endocrine and blood cells. Our newly developed method, the two-photon extracellular polar-tracer imaging-based quantification (TEPIQ) method, can be used for determining fusion pores and the diameters of vesicles smaller than the diffraction-limited resolution. Furthermore, two-photon microscopy has the demonstrated capability of obtaining cross-sectional images from deep layers within nearly intact tissue samples over long observation times with excellent spatial resolution. Recently, we have successfully observed a neuron located deeper than 0.9 mm from the brain cortex surface in an anesthetized mouse. This microscopy also enables the monitoring of long-term changes in neural or glial cells in a living mouse. This minireview describes both the current and anticipated capabilities of two-photon microscopy, based on a discussion of previous publications and recently obtained data.

• 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.

• 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.

• 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.

• Medical Lasers
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• v.10 no.1
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• pp.1-6
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• 2021

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.

• 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.

• 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.

• 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.

• Bulletin of the Korean Chemical Society
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• v.32 no.spc8
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• pp.2889-2890
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• 2011

• Electronics and Telecommunications Trends
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• v.36 no.5
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• pp.1-8
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• 2021

Over the last three decades, the development of Ti:sapphire femtosecond lasers has led to advancements in scientific and industrial fields. In particular, these advanced lasers show great potential for applications with bio-imaging and medical surgery, such as two-photon microscopy, nonlinear Raman microscopy, optical coherence tomography, and ophthalmic surgery. Herein, we present a detailed description of the theoretical and experimental physics of Kerr-lens mode-locked femtosecond Ti:sapphire lasers and its two-photon microscopy.