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. Developmental Dynamics 235:2940 -2948, 2006.

Research in the life sciences increasingly involves the investigation of fast dynamic processes at the cellular and subcellular level. It requires tools to image complex systems with high temporal resolution in three-dimensional space. For this task, we introduce the concept of a fast fluorescence line scanner providing image acquisition speeds in excess of 100 frames per second at 512 x 512 pixels. Because the system preserves the capability for optical sectioning of confocal systems, it allows us to observe processes with three-dimensional resolution. We describe the principle of operation, the optical characteristics of the microscope, and cover several applications in particular from the field of cell and developmental biology. A commercial system based on the line scanning concept has been realized by Carl Zeiss (LSM 5 LIVE).

The excitation efficiency in two-photon absorption (TPA) microscopy depends strongly - owing to the square dependence of the TPA fluorescence on the excitation intensity - on the temporal width of the excitation pulse. Because of their inherently large frequency bandwidth, ultrashort optical pulses tend to broaden substantially because of dispersion from propagation through the dispersive elements in the microscope. In this paper, the dispersion characteristics of a wide range of microscope objectives are investigated. It is shown that the induced dispersion can be pre-compensated in all cases for pulses as short as 15 fs. Because of the excellent agreement between the results from theoretical modelling and the experimental data, predictions of the possibility of dispersion control for microscope objectives in general, as well as for even shorter pulses, can be inferred. Since for TPA imaging the background due to single photon absorption processes and scattering is independent of the pulse width, proper dispersion pre-compensation - which minimizes the pulse duration at the focal point and hence maximizes the excitation efficiency - provides optimal image contrast in TPA microscopy.

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