[show abstract][hide abstract] ABSTRACT: In this work highly localized femtosecond laser ablation is used to dissect single axons within a living Caenorhabditis elegans (C. elegans). We present a multimodal imaging methodology for the assessment of the collateral damage induced by the laser. This relies on the observation of the tissues surrounding the targeted region using a combination of different high resolution microscopy modalities. We present the use of Second Harmonic Generation (SHG) and Polarization Sensitive SHG (PSHG) to determine damage in the neighbor muscle cells. All the above is done using a single instrument: multimodal microscopy setup that allows simultaneous imaging in the linear and non-linear regimes and femtosecond-laser ablation.
PLoS ONE 01/2013; 8(3):e58600. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: In this study we present for the first time the use of confocal microscopy and laser scanning brightfield microscopy (LSBF) for real time imaging of femtosecond laser nanosurgery and its dynamics in C. elegans. A single multimodal optical workstation that provides the ability to perform femtosecond laser nanosurgery and simultaneous confocal and LSBF imaging was used for the purpose. With this tool several dynamic phenomena concomitant with laser nanosurgery in C. elegans were observed and imaged. Some of these dynamic phenomena, like muscular contraction and single muscle cell stimulation, have been imaged for the first time during nano-neurosurgery of C. elegans.
[show abstract][hide abstract] ABSTRACT: In this work we show that a pulsed laser light placed at a distance is able to modulate the growth of axons of primary neuronal cell cultures. In our experiments continuous wave (CW), chopped CW and modelocked fs (FS) laser light was focused through a microscope objective to a point placed at a distance of about 15 microm from the growth cone. We found that CW light does not produce any significant influence on the axon growth. In contrast, when using pulsed light (chopped CW light or FS pulses), the beam was able to modify the trajectory of the axons, attracting approximately 45% of the observed cases to the beam spot. Such effect could possibly indicate the capacity of neurons to interpret the pulsating NIR light as the source of other nearby cells, resulting in extension of processes in the direction of the source.
Journal of neuroscience methods 11/2009; 186(2):196-201. · 2.30 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
The Review of scientific instruments 08/2009; 80(7):073701. · 1.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: A 3 mW focused femtosecond laser spot at a distance (>15 mum) has shown to attract the fillopodia from growth cones of primary neuronal cell cultures (mice E15). The phenomenological behavior of fillopodia is studied under short durations (~40 min) and different laser light conditions. The analysis of the fillopodia movement showed that they become significantly attracted towards the focused femtosecond laser light. In contrast, the use of continuous wave under the same conditions did not generate the same effect, the results of which were indistinguishable from when there was no laser light present (control condition). These results suggest the possible existence of an optically-induced signaling mechanism in growth cones.
[show abstract][hide abstract] ABSTRACT: In this work, we use a two-photon fluorescence microscope for combined imaging and laser tissue ablation of a living Drosophila Melanogaster embryo. By using tightly focused near-infrared femtosecond pulses at MHz repetition rate and of sub-nanojoule energy we are able to produce microsurgery on the epithelial tissue within a Drosophila embryo at the final stages of its embryonic development. Ablation was performed on labelled and unlabelled embryos during and after dorsal closure. We observed that ablation of GFP-labelled tissue required lower energy deposition than unlabelled tissue ensuring that the tissue ablation is mediated by multiphoton absorption of Green Fluorescent Protein (GFP). In addition, the energy deposition to produce ablation is further decreased during dorsal closure. These results show the presence of additional tensile forces on the tissue during dorsal closure. Furthermore, an increased activity of actin near the laser wounds was observed as the tissue heals.
Journal of Microscopy 12/2008; 232(2):362-8. · 1.63 Impact Factor
[show abstract][hide abstract] ABSTRACT: We report a simple methodology to provide complete pulse characterization at the sample plane of a two-photon excited fluorescence (TPEF) microscope. This is achieved by using backward propagating second-harmonic generation (SHG) from starch granules. Without any modification to the microscope, SHG-autocorrelation traces were obtained by using a single starch granule that was placed alongside the biological specimen being imaged. A spectrally resolved SHG autocorrelation was acquired by placing a spectrometer at the output port of the microscope. Complete in situ pulse information is then directly retrieved in an analytical way using the measurement of electric filed by interferometric spectral trace observation (MEFISTO) technique.
Journal of Microscopy 05/2008; 230(Pt 1):70-5. · 1.63 Impact Factor
[show abstract][hide abstract] ABSTRACT: Dorsal closure is a key morphogenic process that occurs at the last
stages of Drosophila melanogaster embryogenesis. It involves a well
coordinated rearrangement and movement of tissues that resemble
epithelial wound healing in mammals. The cell dynamics and intracellular
signaling pathways that accompany hole closure are expected to be
similar during would healing providing a model system to study
epithelial healing. Here we demonstrate the use of two-photon
fluorescence microscope together with femtosecond laser ablation to
examine the epithelial wound healing during embryonic dorsal closure. By
using tightly focused NIR femtosecond pulses of subnanojoule energy we
are able to produce highly confined microsurgery on the epithelial cells
of a developing embryo. We observed that drosophila epidermis heals from
the laser wounds with increased activity of actin near the wound edges.
[show abstract][hide abstract] ABSTRACT: Second harmonic generation (SHG) imaging has emerged in recent years as an important laboratory imaging technique since it can provide unique structural information with submicron resolution. It enjoys the benefits of non-invasive interaction establishing this imaging modality as ideal for in vivo investigation of tissue architectures. In this study we present, polarization dependant high resolution SHG images of Caenorhabditis elegans muscles in vivo. We imaged a variety of muscular structures such as body walls, pharynx and vulva. By fitting the experimental data into a cylindrical symmetry spatial model we mapped the corresponding signal distribution of the chi(2) tensor and identified its main axis orientation for different sarcomeres of the earth worm. The cylindrical symmetry was considered to arise from the thick filaments architecture of the inside active volume. Moreover, our theoretical analysis allowed calculating the mean orientation of harmonophores (myosin helical pitch). Ultimately, we recorded and analysed vulvae muscle dynamics, where SHG signal decreased during in vivo contraction.
[show abstract][hide abstract] ABSTRACT: Because of its polarization sensitivity, SHG microscopy can provide information about the orientation and degree of structural organization inside biological samples. To fully exploit the above potential, the state of the polarization at the sample plane needs to be known. In this work we present starch granules as a reliable probe for the polarization state of the excitation beam at the sample plane of a high resolution multiphoton microscope. Polarization dependent SHG series of images demonstrated the radial distribution of SHG active molecules inside starch granules. This allowed the granule to exhibit symmetrical SHG emission regions. The pattern rotates along with the rotation of a lambda/2 waveplate and thus, can demonstrate the polarization at the sample plane. Maximum signal in the forward detected geometry appears when imaging starch granules exactly at the hemisphere plane. Symmetric SHG regions rotating with the incoming linear polarization were also recorded in the backward detected geometry. A portion of the backwards detected SHG signal, which corresponds to two rotating equator arcs, does not overlap with the forward SHG signal. Importantly, polarization measurements, performed either in the forward or the backwards directions, have demonstrated the suitability and flexibility of this technique for both detection schemes. As result, observation of the starch signal allowed us to know the polarization of our SHG microscope. Furthermore, by coding this information in an angular representation, we corrected the input values in a theoretical model that predicts the average orientation of SHG active molecules. This has allowed us to map the mean orientation of SHG active molecules in body walls muscle of Caenorhabditis elegans, with pixel resolution.
[show abstract][hide abstract] ABSTRACT: In this paper an ideal approach for pulse characterisation within a multiphoton microscope is demonstrated. This methodology will be used for shaping the pulses with an spatial light modulators (SLM) and to study the influence of arbitrary phase profiles, specifically with large bandwidth pulses, on nonlinear imaging of the biological samples.
Lasers and Electro-Optics, 2007 and the International Quantum Electronics Conference. CLEOE-IQEC 2007. European Conference on; 07/2007
[show abstract][hide abstract] ABSTRACT: Measurable change in the sensory motor machinery of growth cones are induced by non contact femtosecond laser. The focused laser beam with an average power of 3 mW was positioned at some distance away from the closest fillopodia of cortical neurons from primary cell cultures (mice E15). By identifying a set of preliminary parameters we were able to statistically analyze the phenomenological behavior of the fillopodia and classify the effects different conditions of laser light has on the growth cone. Results show that fillopodia become significantly biased towards the focused femtosecond laser light. The same experiment performed with continuous wave (CW) produced results which were indistinguishable from the case where there is no laser light present (placebo condition) indicating no clear effects of the CW laser light on the fillopodia at a distance. These findings show the potential for ultrashort pulsed light to become a new type of pathfinding cue for neuronal growth cones.
[show abstract][hide abstract] ABSTRACT: In this paper, we show that the neuronal fillopodia can detect and respond to the presence of focused femtosecond laser light at a distance, an effect that does not occur when CW illumination is used. Control and manipulation of axonal growth is of extreme importance in the field of neurosciences as it could lead to advances such as in-vivo nerve regeneration or arbitrary patterning of neural circuits.