Simultaneous Two- and Three-Photon Deep Imaging of Autofluorescence in Bacterial Communities.

The intrinsic fluorescence of bacterial samples has a proven potential for label-free bacterial characterization, monitoring bacterial metabolic functions, and as a mechanism for tracking the transport of relevant components through vesicles. The reduced scattering and axial confinement of the excitation offered by multiphoton imaging can be used to overcome some of the limitations of single-photon excitation (e.g., scattering and out-of-plane photobleaching) to the imaging of bacterial communities. In this work, we demonstrate in vivo multi-photon microscopy imaging of Streptomyces bacterial communities, based on the excitation of blue endogenous fluorophores, using an ultrafast Yb-fiber laser amplifier. Its parameters, such as the pulse energy, duration, wavelength, and repetition rate, enable in vivo multicolor imaging with a single source through the simultaneous two- and three-photon excitation of different fluorophores. Three-photon excitation at 1040 nm allows fluorophores with blue and green emission spectra to be addressed (and their corresponding ultraviolet and blue single-photon excitation wavelengths, respectively), and two-photon excitation at the same wavelength allows fluorophores with yellow, orange, or red emission spectra to be addressed (and their corresponding green, yellow, and orange single-photon excitation wavelengths). We demonstrate that three-photon excitation allows imaging over a depth range of more than 6 effective attenuation lengths to take place, corresponding to an 800 micrometer depth of imaging, in samples with a high density of fluorescent structures.

Non-Destructive Direct Pericarp Thickness Measurement of Sorghum Kernels Using Extended-Focus Optical Coherence Microscopy.

Non-destructive measurements of internal morphological structures in plant materials such as seeds are of high interest in agricultural research. The estimation of pericarp thickness is important to understand the grain quality and storage stability of seeds and can play a crucial role in improving crop yield. In this study, we demonstrate the applicability of fiber-based Bessel beam Fourier domain (FD) optical coherence microscopy (OCM) with a nearly constant high lateral resolution maintained at over ~400 µm for direct non-invasive measurement of the pericarp thickness of two different sorghum genotypes. Whereas measurements based on axial profiles need additional knowledge of the pericarp refractive index, en-face views allow for direct distance measurements. We directly determine pericarp thickness from lateral sections with a 3 µm resolution by taking the width of the signal corresponding to the pericarp at the 1/e threshold. These measurements enable differentiation of the two genotypes with 100% accuracy. We find that trading image resolution for acquisition speed and view size reduces the classification accuracy. Average pericarp thicknesses of 74 µm (thick phenotype) and 43 µm (thin phenotype) are obtained from high-resolution lateral sections, and are in good agreement with previously reported measurements of the same genotypes. Extracting the morphological features of plant seeds using Bessel beam FD-OCM is expected to provide valuable information to the food processing industry and plant breeding programs.

Extended focal depth Fourier domain optical coherence microscopy with a Bessel-beam - LP02 mode - from a higher order mode fiber.

We present a robust fiber-based setup for Bessel-like beam extended depth-of-focus Fourier-domain optical coherence microscopy, where the Bessel-like beam is generated in a higher order mode fiber module. In this module a stable guided LP02 core mode is selectively excited by a long period grating written in the higher order mode fiber. Imaging performance of this system in terms of lateral resolution and depth of focus was analyzed using samples of suspended microbeads and compared to the case where illumination is provided by the fundamental LP01 mode of a single mode fiber. Illumination with the LP02 mode allowed for a lateral resolution down to 2.5 µm as compared to 4.5 µm achieved with the LP01 mode of the single mode fiber. A three-fold enhancement of the depth of focus compared to a Gaussian beam with equally tight focus is achieved with the LP02 mode. Analysis of the theoretical lateral point spread functions for the case of LP01 and LP02 illumination agrees well with the experimental data. As the design space of waveguides and long-period gratings allows for further optimization of the beam parameters of the generated Bessel-like beams in an all-fiber module, this approach offers a robust and yet flexible alternative to free-space optics approaches or the use of conical fiber tips.

Fast volumetric calcium imaging across multiple cortical layers using sculpted light.

Although whole-organism calcium imaging in small and semi-transparent animals has been demonstrated, capturing the functional dynamics of large-scale neuronal circuits in awake behaving mammals at high speed and resolution has remained one of the main frontiers in systems neuroscience. Here we present a method based on light sculpting that enables unbiased single- and dual-plane high-speed (up to 160 Hz) calcium imaging as well as in vivo volumetric calcium imaging of a mouse cortical column (0.5 mm × 0.5 mm × 0.5 mm) at single-cell resolution and fast volume rates (3-6 Hz). We achieved this by tailoring the point-spread function of our microscope to the structures of interest while maximizing the signal-to-noise ratio using a home-built fiber laser amplifier with pulses that are synchronized to the imaging voxel speed. This enabled in vivo recording of calcium dynamics of several thousand neurons across cortical layers and in the hippocampus of awake behaving mice.

Optical detection of attosecond ionization induced by a few-cycle laser field in a transparent dielectric material.

We observe an optical signature induced by the modulation of electron density inside a bulk transparent solid that is quasiperiodically ionized on an attosecond time scale by electric field peaks of a focused few-cycle laser pulse. The emitted optical signal resulting from the attosecond ionization dynamics is spatially, temporally and spectrally isolated from concomitant optical responses through the use of a noncollinear pump-probe technique. The method holds promise for developing an attosecond metrology for bulk solids, in which, unlike in the established attosecond metrology of gases and surfaces, direct detection of charged particles is unfeasible.

Optical detection of tunneling ionization.

We have experimentally detected optical harmonics that are generated due to a tunneling-ionization-induced modulation of the electron density. The optical signature of electron tunneling can be isolated from concomitant optical responses by using a noncollinear pump-probe setup. Whereas previously demonstrated tools for attosecond metrology of gases, plasmas, and surfaces rely on direct detection of charged particles, detection of the background-free time-resolved optical signal, which uniquely originates from electron tunneling, offers an interesting alternative that is especially suited for systems in which free electrons cannot be directly measured.

Self-compression of millijoule 1.5 microm pulses.

We demonstrate a four-stage optical parametric chirped-pulse amplification system that delivers carrier-envelope phase-stable approximately 1.5 microm pulses with energies up to 12.5 mJ before recompression. The system is based on a fusion of femtosecond diode-pumped solid-state Yb technology and a picosecond 100 mJ Nd:YAG pump laser. Pulses with 62 nm bandwidth are recompressed to a 74.4 fs duration close to the transform limit. To show the way toward a terawatt-peak-power single-cycle IR source, we demonstrate self-compression of 2.2 mJ pulses down to 19.8 fs duration in a single filament in argon with a 1.5 mJ output energy and 66% energy throughput.

Plasma-blueshift spectral shear interferometry for characterization of ultimately short optical pulses.

We introduce a bandwidth-unlimited, dispersion- and shear-self-calibrated, timing-jitter-free pulse measurement technique based on a quasi-linear temporal phase modulation in a gas weakly ionized by a long pump pulse. Results of a 5 fs pulse characterization are reported.

Few-cycle carrier envelope phase-dependent stereo detection of electrons.

The spatial distribution of electrons emitted from atoms by few-cycle optical fields is known to be dependent on the carrier envelope phase, i.e., the phase of the field with respect to the pulse envelope. With respect to Paulus et al. [Phys. Rev. Lett.91, 253004 (2003)] we propose a greatly simplified device to measure and control the carrier envelope phase of few-cycle pulses with an accuracy of better than pi/10 based on this principle. We compared different schemes to control the carrier envelope phase of our pulses.