David Artigas

In vivo, pixel-resolution mapping of thick filaments' orientation in nonfibrilar muscle using polarization-sensitive second harmonic generation microscopy

The polarization dependence of second harmonic generation (SHG) microscopy is used to uncover structural information in different muscle cells in a living Caenorhabditis elegans (C. elegans) nematode. This is done by using a generalized biophysical model in which element ratios for the associated second-order nonlinear tensor and angular orientations for thick filaments are retrieved using a pixel-by-pixel fitting algorithm. As a result, multiple arbitrary orientations of thick filaments, at the pixel-resolution level, are revealed in the same image. The validity of our method is first corroborated in well-organized thick filaments such as the nonfibrilar body wall muscles. Next, a region of the nonstriated muscular cells of the pharynx is analyzed by showing different regions with homogenous orientations of thick filament as well as their radial distribution. As a result, different sets of the nonstriated muscle cell groups in the pharynx of this nematode were exposed. This methodology is presented as a filtering mechanism to uncover biological information unreachable by common intensity SHG microscopy. Finally, a method to experimentally retrieve the distribution of the effective orientation of active SHG molecules is proposed and tested.

Ultrashort pulse characterisation with SHG collinear-FROG

We outline criteria for fast and accurate acquisition of collinear FROG (CFROG) trace and how it can be transformed into the more traditional noncollinear FROG trace. The CFROG has an intrinsically simple geometry that provides greater versatility as well as the ability for built-in delay calibration and enhanced error-checking. The procedure, based on data processing, allows conventional SHG-FROG retrieval algorithms to be used. This technique is tested numerically and experimentally giving excellent results. This work represents an attractive alternative to the traditional, more complex non-collinear FROG technique while, at the same time, extending its use to experiments where collinear geometry is imposed.

Compact ultrafast semiconductor disk laser: targeting GFP based nonlinear applications in living organisms

We present a portable ultrafast Semiconductor Disk Laser (SDL) (or vertical extended cavity surface emitting laser—VECSELs), to be used for nonlinear microscopy. The SDL is modelocked using a quantum-dot semiconductor saturable absorber mirror (SESAM), delivering an average output power of 287 mW, with 1.5 ps pulses at 500 MHz and a central wavelength of 965 nm. Specifically, despite the fact of having long pulses and high repetition rates, we demonstrate the potential of this laser for Two-Photon Excited Fluorescence (TPEF) imaging of in vivo Caenorhabditis elegans (C. elegans) expressing Green Fluorescent Protein (GFP) in a set of neuronal processes and cell bodies. Efficient TPEF imaging is achieved due to the fact that this wavelength matches the peak of the two-photon action cross section of this widely used fluorescent marker. The SDL extended versatility is shown by presenting Second Harmonic Generation images of pharynx, uterus, body wall muscles and its potential to be used to excite other different commercial dyes. Importantly this non-expensive, turn-key, compact laser system could be used as a platform to develop portable nonlinear bio-imaging devices.

A simple scanless two-photon fluorescence microscope using selective plane illumination

We demonstrate a simple scanless two-photon (2p) excited fluorescence microscope based on selective plane illumination microscopy (SPIM). Optical sectioning capability is presented and depth-resolved imaging of cameleon protein in C. elegans pharyngeal muscle is implemented.

Lossless directional guiding of light in dielectric nanosheets using Dyakonov surface waves

Guiding light at the nanoscale is usually accomplished using surface plasmons. However, plasmons propagating at the surface of a metal sustain propagation losses. A different type of surface excitation is the Dyakonov surface wave. These waves, which exist in lossless media, were predicted more than two decades ago but observed only recently. Dyakonov surface waves exist when at least one of the two media forming the surface exhibits a suitable anisotropy of refractive indexes. Although propagating only within a narrow range of directions, these waves can be used to create modes supported by ultrathin films that confine light efficiently within film thicknesses well below the cutoff thickness required in standard waveguides. Here, we show that 10 nm and 20 nm dielectric nanosheets of aluminium oxide clad between an anisotropic crystal (lithium triborate) and different liquids support Dyakonov-like modes. The direction of light propagation can be controlled by modulating the refractive index of the cladding. The possibility of guiding light in nanometre-thick films with no losses and high directionality makes Dyakonov wave modes attractive for planar photonic devices in schemes similar to those currently employing long-range plasmons.

Measurement and correction of in vivo sample aberrations employing a nonlinear guide-star in two-photon excited fluorescence microscopy

We demonstrate that sample induced aberrations can be measured in a nonlinear microscope. This uses the fact that two-photon excited fluorescence naturally produces a localized point source inside the sample: the nonlinear guide-star (NL-GS). The wavefront emitted from the NL-GS can then be recorded using a Shack-Hartmann sensor. Compensation of the recorded sample aberrations is performed by the deformable mirror in a single-step. This technique is applied to fixed and in vivo biological samples, showing, in some cases, more than one order of magnitude improvement in the total collected signal intensity.

Image formation by linear and nonlinear digital scanned light-sheet fluorescence microscopy with Gaussian and Bessel beam profiles

We present the implementation of a combined digital scanned light-sheet microscope (DSLM) able to work in the linear and nonlinear regimes under either Gaussian or Bessel beam excitation schemes. A complete characterization of the setup is performed and a comparison of the performance of each DSLM imaging modality is presented using in vivo Caenorhabditis elegans samples. We found that the use of Bessel beam nonlinear excitation results in better image contrast over a wider field of view.

Measurement of electric field by interferometric spectral trace observation.

We present a new methodology that obtains, in an analytical way, the complex electric field of ultrashort pulses. This methodology is based only on Fourier analysis of the frequency components of spectrally resolved interferometric collinear autocorrelations. We present an experimental demonstration of this technique and the results are compared with the conventional second-harmonic generation frequency-resolved optical gating technique.

Quantitative discrimination between endogenous SHG sources in mammalian tissue, based on their polarization response

In this study, the second harmonic generation (SHG) response to polarization and subsequent data analysis is used to discriminate, in the same image, different SHG source architectures with pixel resolution. This is demonstrated in a mammalian tissue containing both skeletal muscle and fibrilar collagen. The SHG intensity variation with the input polarization (PSHG) is fitted pixel by pixel in the image using an algorithm based on a generalized biophysical model. The analysis provides the effective orientation, theta(e), of the different SHG active structures (harmonophores) at every pixel. This results in a new image in which collagen and muscle are clearly differentiated. In order to quantify the SHG response, the distribution of theta(e) for every harmonophore is obtained. We found that for collagen, the distribution was centered at theta(e) = 42.7 degrees with a full width at half maximum of theta = 5.9 degrees while for muscle theta(e) = 65.3 degrees , with theta = 7.7 degrees . By comparing these distributions, a quantitative measurement of the discrimination procedure is provided.

Asymmetrical splitting of higher-order optical solitons induced by quintic nonlinearity

We address the effect of quintic nonlinearities in the propagation of optical solitons in cubic nonlinear media. Our focus is on the decay of higher-order solitons in the presence of self-defocusing quintic perturbations. We show that, in spite of the fact that the governing evolution equations are symmetric, the quintic nonlinearity produces the asymmetrical self-splitting of the solitons. We study in detail the self-splitting process and compare the results with the soliton evolution with self-focusing quintic perturbations.