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 λ/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.
KEYWORDS: Second-harmonic generation, Polarization, In vivo imaging, Image resolution, Signal detection, Harmonic generation, Tissues, Collagen, Nonlinear optics, Data modeling
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 χ(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.
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.
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.
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