Multiphoton microscopy for observation of living cells

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Laser scanning confocal microscopes have been taking important roles in biology with its high spatial resolution in three dimensions allowing us to obtain clear three-dimensional images of cells and tissues. As a further improvement of laser microscopy, multiphoton microscopes are expected to be promising tools for observing living cell and tissues. Multiphoton process used in the multiphoton microscopes provide several benefits in optical microscopes, such as higher spatial resolution in three dimensions, higher image contrast, reduction of phototoxicity, and capability of imaging cell functions. Those benefits in multiphoton microscope are derived from optical nonlinear phenomena induced in a tightly focused laser light in a specimen. Two-photon fluorescence excitation is the most popular nonlinear phenomenon used for microscopy and have already been used in commercial laser scanning microscope systems. Second-harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) are also the nonlinear phenomena that have been applied for laser microscopes. SHG is a conversion effect of light wavelength that gives a half wavelength of light incident to a material. In a SHG microscope, a laser beam is focused into a specimen, and light possessing a half wavelength of the incident is generated from the focal point. Detecting this generated light with scanning the focal spot gives an image of the specimen. Since the wavelength of signal light is half of the incident laser light and its intensity is proportional to the squared intensity of the incident light, the benefits in the use of SHG is quite similar to those in two-photon fluorescence microscopes. However, SHG has strong sensitivity to molecular orientation of dye molecules. Strong SHG signal can be obtained at positions consist of relatively well-ordered molecules, such as cell membrane and collagen fibers. In case of membrane imaging, it is known that the molecular condition is affected by membrane potential of a cell. The use of this effect has been proposed for measuring membrane potential with high sensitivity. Molecular information in cells or tissues can also be obtained by using a CARS microscope. CARS signal carries information of vibrational property of molecules (similar to infrared spectroscopy). In the CARS microscope, two coherent laser beams with different wavelength are incident to a specimen and tuning the difference of two wavelength to vibration energy of molecules gives strong CARS signal emission from the specimen. Since the CARS microscope monitors the vibrational state of the molecules, images of the specimen can be obtained without staining with dye molecules. This is the most important advantage in the CARS microscope because staining cells with fluorescent dye gives undesired cell condition and degrade cell viability. In demonstration of the CARS microscope, living HeLa cells are clearly observed by tuning the laser light to aliphatic CH vibration that are rich in mitochondria. For inducing those nonlinear phenomena, a near-infrared (NIR) pulse laser is used. Since NIR light has almost no single-photon absorption in living cells and tissues and the multiphoton process can be induced only in a focal volume of the laser light, undesired phototoxicity can be dramatically reduced. For example, in case of a single photon fluorescence microscope, a whole illuminated are in the specimen has possibility to be damaged. This is also the one of most important advantages in multiphoton microscopy.
日本組織細胞化学会の論文

Multiphoton microscopy for observation of living cells

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