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.

Controlling and monitoring temperature at the single cell level has become pivotal in biology and medicine. Indeed, temperature influences many intracellular processes and is also involved as an activator in novel therapies. Aiming to assist such developments, several approaches have recently been proposed to probe cell temperature in vitro. None of them have so far been extended to a living organism. Here we present the first in vivo intracellular temperature imaging. Our technique relies on measuring the fluorescence polarization anisotropy of green fluorescent protein (GFP) on a set of GFP expressing neurons in Caenorhabditis elegans (C. elegans). We demonstrate fast and noninvasive monitoring of subdegree temperature changes on a single neuron induced by local photoheating of gold nanoparticles. This simple and biocompatible technique is envisioned to benefit several fields including hyperthermia treatment, selective drug delivery, thermal regulation of gene expression and neuron laser ablation.

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.

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.

Abstract. Live microscopy techniques ͑i.e., differential interference contrast, confocal microscopy, etc.͒ have enabled the understanding of the mechanisms involved in cells and tissue formation. In long-term studies, special care must be taken in order to avoid sample damage, restricting the applicability of the different microscopy techniques. We demonstrate the potential of using third-harmonic generation ͑THG͒ microscopy for morphogenesis/embryogenesis studies in living Caenorhabditis elegans ͑C. elegans͒. Moreover, we show that the THG signal is obtained in all the embryo development stages, showing different tissue/structure information. For this research, we employ a 1550-nm femtosecond fiber laser and demonstrate that the expected water absorption at this wavelength does not severely compromise sample viability. Additionally, this has the important advantage that the THG signal is emitted at visible wavelengths ͑516 nm͒. Therefore, standard collection optics and detectors operating near maximum efficiency enable an optimal signal reconstruction. All this, to the best of our knowledge, demonstrates for the first time the noninvasiveness and strong potential of this particular wavelength to be used for highresolution four-dimensional imaging of embryogenesis using unstained C. elegans in vivo samples. © 2010 Society of Photo-Optical Instrumentation Engineers.

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In this work we propose and build a multimodal optical workstation that extends a commercially available confocal microscope (Nikon Confocal C1-Si) to include nonlinear/multiphoton microscopy and optical manipulation/stimulation tools such as nanosurgery. The setup allows both subsystems (confocal and nonlinear) to work independently and simultaneously. The workstation enables, for instance, nanosurgery along with simultaneous confocal and brightfield imaging. The nonlinear microscopy capabilities are added around the commercial confocal microscope by exploiting all the flexibility offered by this microscope and without need for any mechanical or electronic modification of the confocal microscope systems. As an example, the standard differential interference contrast condenser and diascopic detector in the confocal microscope are readily used as a forward detection mount for second harmonic generation imaging. The various capabilities of this workstation, as applied directly to biology, are demonstrated using the model organism Caenorhabditis elegans.

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