Development of a Multiphoton Microscope for Observation of Ca^<2+> Dynamics at 500μm Depth in a Heart with 1ms Temporal Resoki

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We developed a real-time two-photon confocal fluorescence microscope which enables us to observe dynamics of living specimens at video rate. In the developed microscope, a microlens-and pinhole-array disk are used for simultaneous excitation of fluorescence and confocal fluorescence detection in the specimen. The rotation of the disks scans the specimen in about 3ms, and the fluorescence images can be obtained by a intensified CCD came. Depending on the image acquisition rate of the CCD camera, the temporal resolution can be enhanced up to 3ms. We observed Ca ion dynamics in a rat whole-heart by using the developed microscope to demonstrate its imaging properties. A rat whole-heart was loaded with a Ca ion indicator, fluo-4AM, and a Tyrode solution through Langendorf perfusion for 30 minutes before the observation. We used a mode-locked Ti : Sapphire laser as a light source for excitation of two-photon fluorescence, and an NA 0.9 water immersion objective lens for illumination and observation of the specimen. The heart was also perfused with the solution during the observation. Sudden rise of Ca ion concentration (Ca ion transient) and propagation of high concentrated Ca ion wave can be observed by the developed microscope.We also investigated the limitation of the observation depth of the developed microscope. The scattering efficiency of the specimen was estimated by measuring size of a fluorescent spot from different depth in a rat whole-heart loaded with fluo-4/AM.In this experiment, we found that the size of the focus is widened with the observation depth exponentially, which reduces the amount of detectable fluorescence at the detector. We also measured fluorescence intensity from the different observation depth in the same specimen. From above two experiments, the maximum observation depth was estimated to be about 100μm in a case of observing a rat-whole heart.