Real-Time Two-Photon Microscopy and Its Application for In Situ Imaging.

Abstract

The recent developments in fluorescentprobes and laser technologies have provided us opportunities to observe the biological functions in living organs and tissues under a microscope. Although single-photon excitation confocal microscopy is an indispensable tool for observing these specimens, the observable depth of this microscopy is still not enough to analyze the functions of whole parts of organs in living organisms. To observe the deeper parts, we have attempted to develop new real-time two-photon excitation microscopy equipped with a microlens-arrayed multipinhole scanning device. Using this real-time twophoton microscope, we examined the dynamics of the intracellular calcium concentration ([Ca 2+ ] i ) in the cardiac myocytes in the rat whole heart to understand their physiological functions and mechanisms. We could observe Ca 2+ waves on the surface in the whole heart. However, we still need higher-energy laser power for twophoton excitation and visualization of [Ca 2+ ] i dynamics in the deeper parts of the whole heart.

Spatiotemporal Visualization of Intracellular Ca2+ in Living Heart Muscle Cells Viewed by Confocal Laser Scanning Microscopy.

“…Namely, this approach would be highly applicable to other diseased hearts, such as those under hypertrophy or those injured by ischemia/reperfusion [60], genetically engineered hearts. If the limitations of this technique are overcome, such as toxicity of excitation laser, time-dependent compartmentalization of the fluorescence indicators [21], and difficulties in observations within deeper areas [20,28], this method will surely provide greater insight into understanding of [Ca 2+ ]i dynamics within the heart.…”

Imaging Approaches and the Quantitative Analysis of Heart Development

“…As an alternative, two-photon microscopy employs an excitation laser with twice the wavelength of the desired excitation (such as the one used in conventional singlephoton confocal), which reduces light scattering within a sample by up to 1/16 [49]. This method localizes the fluorescence excitation at the focal point of interest, allowing for three-dimensional resolution, similar to that of a conventional confocal laser scanning microscope, while also minimizing phototoxicity on out-of-focus planes and improving penetration, thanks to the longer wavelength.…”