A key challenge when imaging living cells is how to noninvasively extract the most spatiotemporal information possible. Unlike popular wide-field and confocal methods, plane-illumination microscopy limits excitation to the information-rich vicinity of the focal plane, providing effective optical sectioning and high speed while minimizing out-of-focus background and premature photobleaching. Here we used scanned Bessel beams in conjunction with structured illumination and/or two-photon excitation to create thinner light sheets (<0.5 μm) better suited to three-dimensional (3D) subcellular imaging. As demonstrated by imaging the dynamics of mitochondria, filopodia, membrane ruffles, intracellular vesicles and mitotic chromosomes in live cells, the microscope currently offers 3D isotropic resolution down to ~0.3 μm, speeds up to nearly 200 image planes per second and the ability to noninvasively acquire hundreds of 3D data volumes from single living cells encompassing tens of thousands of image frames.

We demonstrate the highest intensity - 300 TW laser by developing booster amplifying stage to the 50-TW-Ti:sapphire laser (HERCULES). To our knowledge this is the first multi-100TW-scale laser at 0.1 Hz repetition rate.

We generated a record peak intensity of 0.7 x 10(22) W/cm2 by focusing a 45-TW laser beam with an f/0.6 off-axis paraboloid. The aberrations of the paraboloid and the low-energy reference laser beam were measured and corrected, and a focal spot size of 0.8 microm was achieved. It is shown that the peak intensity can be increased to 1.0 x 10(22) W/cm2 by correction of the wave front of a 45-TW beam relative to the reference beam. The phase and amplitude measurement provides for an efficient full characterization of the focal field.

Despite all the advances in nonlinear microscopy, all existing instruments are constrained to obtain images of one focal plane at a time. In this Letter we demonstrate a two-photon absorption fluorescence scanning microscope capable of imaging two focal planes simultaneously. This is accomplished by temporally demultiplexing the signal coming from two focal volumes at different sample depths. The scheme can be extended to three or more focal planes.

A quantitative understanding of tissue morphogenesis requires description of the movements of individual cells in space and over time. In transparent embryos, such as C. elegans, fluorescently labeled nuclei can be imaged in three-dimensional time-lapse (4D) movies and automatically tracked through early cleavage divisions up to ~350 nuclei. A similar analysis of later stages of C. elegans development has been challenging owing to the increased error rates of automated tracking of large numbers of densely packed nuclei. We present Nucleitracker4D, a freely available software solution for tracking nuclei in complex embryos that integrates automated tracking of nuclei in local searches with manual curation. Using these methods, we have been able to track >99% of all nuclei generated in the C. elegans embryo. Our analysis reveals that ventral enclosure of the epidermis is accompanied by complex coordinated migration of the neuronal substrate. We can efficiently track large numbers of migrating nuclei in 4D movies of zebrafish cardiac morphogenesis, suggesting that this approach is generally useful in situations in which the number, packing or dynamics of nuclei present challenges for automated tracking.

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We demonstrate the use of a simple tool to simultaneously visualize and characterize chromatic and spherical aberrations that are present in multiphoton microscopy. Using two-dimensional Fourier transform spectral interferometry, we measured these aberrations, deducing in a single shot spatiotemporal effects in high-numerical-aperture objectives.

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