Katsumasa Fujita

Generation of calcium waves in living cells by pulsed-laser-induced photodisruption

Here, we present an optical technique that can induce waves of calcium ion concentration in live biological cells. Ca2+ waves were induced by femtosecond pulsed-laser illumination. Living HeLa cells were exposed to focused 140 fs pulses of 780 nm wavelength at 30 mW average power. Ca2+ waves were imaged by fluorescence and were observed to propagate from the laser focal point inside the cell. Photoinduced generation of Ca2+ waves can be performed at any point inside the cell, an improvement over previous mechanical or biochemical stimulation techniques.

In situ visualization of the intracellular Ca2+ dynamics at the border of the acute myocardial infarct.

Ischemic insult to the heart produces myocyte Ca2+ ([Ca2+]i) overload. However, little is known about spatiotemporal changes in [Ca2+]i within the ischemic heart in situ at the cellular level. Using real-time confocal microscopy, we successfully visualized [Ca2+]i dynamics at the border zone on the subepicardial myocardium of the heart 2 h after coronary ligations followed by loading with fluo 3/AM. Three distinct regions were identified in the acute infarcted heart. In intact regions, the myocytes showed spatially uniform Ca2+ transients synchronously to QRS complex in the electrocardiogram. The myocytes at the infarcted regions showed no fluorescence intensity (FI). At the border zones between the intact and infarcted regions, Ca2+ waves emerged sporadically and randomly, instead of Ca2+ transients, at a mean frequency of 11.5 ± 8.5 min/cell with a propagation velocity of 151.0 ± 35.7 μm/sec along the longitudinal axis of the individual myocytes. In addition, some myocytes within the border zone exhibited homogeneously high static FI, indicating severe Ca2+ overload. In summary, we provided the first direct evidence of abnormal [Ca2+]i dynamics in acute infarcted hearts at the cellular level. The observed diversity in spatiotemporal [Ca2+]i dynamics at the border zone may contribute to the arrhythmias or contractile failure in acute myocardial infarction.

Three-dimensional subsurface microprocessing of collagen by ultrashort laser pulses

Submicron sized damage spots were created by multiphoton absorption of ultrashort 775 nm laser pulses in collagen gel stained with fluorescent dye. Submicron nodules were formed in the collagen both near the surface and at a depth of up to 30 μm. We have shown that large numbers of pulses at low pulse energy provide well-controlled sample damage restricted to a small focal volume. The damage threshold was dependent on dye concentration but not significantly dependent on the depth. This will be useful for experiments that study laser-induced damage in living biological samples on a cellular scale and deep within the specimen.