Biphoton focusing for two-photon excitation

We study two-photon excitation using biphotons generated via the process of spontaneous parametric down-conversion in a nonlinear crystal. We show that the focusing of these biphotons yields an excitation distribution that is essentially the same as the distribution of one-photon excitation at the pump wavelength. We also demonstrate that biphoton excitation in the image region yields a distribution whose axial width is approximately that of the crystal thickness and whose transverse width is that of the pump at the input to the crystal.Comment: Accepted for publication in Physical Review

Rapid three-dimensional imaging and analysis of the beating embryonic heart reveals functional changes during development

We report an accurate method for studying the functional dynamics of the beating embryonic zebrafish heart. The fast cardiac contraction rate and the high velocity of blood cells have made it difficult to study cellular and subcellular events relating to heart function in vivo. We have devised a dynamic three-dimensional acquisition, reconstruction, and analysis procedure by combining (1) a newly developed confocal slit-scanning microscope, (2) novel strategies for collecting and synchronizing cyclic image sequences to build volumes with high temporal and spatial resolution over the entire depth of the beating heart, and (3) data analysis and reduction protocols for the systematic extraction of quantitative information to describe phenotype and function. We have used this approach to characterize blood flow and heart efficiency by imaging fluorescent protein-expressing blood and endocardial cells as the heart develops from a tube to a multichambered organ. The methods are sufficiently robust to image tissues within the heart at cellular resolution over a wide range of ages, even when motion patterns are only quasiperiodic. These tools are generalizable to imaging and analyzing other cyclically moving structures at microscopic scales