Thomas A. Planchon

Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination

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.

Simultaneous imaging of multiple focal planes using a two-photon scanning microscope

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.

Quantitative semi-automated analysis of morphogenesis with single-cell resolution in complex embryos

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.

Simultaneous visualization of spatial and chromatic aberrations by two-dimensional Fourier transform spectral interferometry.

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.

Measurement of pump-induced transient lensing in a cryogenically-cooled high average power Ti:sapphire amplifier

The transient thermal lensing in a liquid-nitrogren cooled kilohertz multipass amplifier is quantitatively measured with spatially-resolved Fourier transform spectral interferometry. A pump-probe arrangement allows the observation of a polarization-dependent non-thermal component following the fluorescence timescale: additional cooling would not suppress this residual lensing. We also observe a time-dependent thermal component that has a timescale sufficiently fast to indicate that there is cooling between shots even at a repetition rate of 1 kHz. The value of pump-induced lensing would be underestimated when performing time-averaged measurements of pump-induced phase shifts.

Complete characterization of a spatiotemporal pulse shaper with two-dimensional Fourier transform spectral interferometry

Spatiotemporal pulse shaping is characterized with two-dimensional Fourier transform spectral interferometry. A deformable-mirror-based bidimensional pulse shaper is used to create simple spatiotemporal structures on a femtosecond pulse, structures that are directly calculated from the measured spatiospectral phases and intensities.

Characterization of coupled nonlinear spatiospectral phase following an ultrafast self-focusing interaction

We use collinear spatially resolved spectral interferometery to characterize the nonlinear phase changes experienced by an intense ultrashort pulse propagating in glass. The measurement yields the spectrally dependent wavefront, allowing us to measure the spatial and chromatic aberrations of the nonlinearly induced lens. For these conditions, we find that while the shape of the spatial wavefront follows the beam profile as expected, the spectral dependence of the lensing power is determined by the self-phase modulation. The simultaneous measurement of the nonlinear spatiospectral phase demonstrates how the nonlinear spectral phase is coupled to self-focusing.

Spatiotemporal dynamics of cross-polarized wave generation

We use Spatially and Spectrally Resolved Interferometry (SSRI) to investigate cross-polarized wave (XPW) generation. We find that the XPW pulse is √3 smaller than the input in the spatiotemporal domain regardless of input chirp.

Adaptive correction of a tightly focused, high-intensity laser beam by use of a third-harmonic signal generated at an interface

By using the third-harmonic signal generated at an air-dielectric interface, we demonstrate a novel way of correcting wavefront aberrations induced by high-numerical-aperture optics. The third harmonic is used as the input physical parameter of a genetic algorithm working in closed loop with a 37-actuator deformable mirror. This method is simple and reliable and can be used to correct aberrations of tightly focused beams, a regime where other methods have limitations. Improvement of the third-harmonic signal generated with an f/1.2 parabolic mirror by 1 order of magnitude is demonstrated.

Wavefront correction and aberrations pre-compensation in the middle of Petawatt-class CPA laser chains

We describe preliminary experiences to validate correction of wavefront aberrations in middle of laser chain. This technique allows correction of aberrations from first part, and the pre-compensation of aberrations built in second part of laser.