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.
It is well known that the efficiency and selectivity of two-photon excited fluorescence (TPEF) process can
depend on various parameters of the ultrashort pulses, such as the pulse intensity and phase, which interact with the
specimen. In order to completely understand this dependence and to obtain optimal TPEF images, techniques like
Collinear Frequency Resolved Optical Gating (CFROG) arrangement can be implemented in a microscope for complete
pulse characterisation at the sample plane. However, this adds complexity that that additional forward collecting optics is
required as well as a suitable frequency doubling crystal.
Here we report a simple way to characterize the pulses within a multiphoton microscope that do not require
forward collecting optics. This is achieved by taking advantage of the fact that backward propagating second harmonic
generation (SHG) signal can be easily generated from starch granules. Since both the fluorescence and SHG signals can
be collected using the same detection scheme the experimental arrangement is considerably simplified. Starch, being a
non- toxic and non-soluble material does not affect living cells allowing the pulse characteristics to be measured <i>in situ</i>,
without the need to move the sample.
We obtained real-time SHG-autocorrelations traces by using a single starch granule that was placed alongside
the living HeLa cells (GFP labeled) being imaged. Furthermore by placing a spectrometer at the output port of the
microscope, a spectrally resolved SHG autocorrelation was acquired allowing complete characterisation of the pulse to
be carried out. The temporal intensity and phase profile were retrieved using CFROG technique. Marginal analysis was
carried out to ensure that the experimental data was successfully acquired.
We investigate the local field spectroscopy of gold dimers by Two-Photon Photoluminescence (TPL) microscopy. A direct comparison with far-field scattering measurements shows that TPL provides additional data on the structure modes of major importance for their use in SERS, enhanced fluorescence and sensing.
In this paper we report the use of a starch as a non-linear medium for characterising ultrashort pulses. The starch suspension in water is sandwiched between a slide holder and a cover-slip and placed within the sample plane of the nonlinear microscope. This simple arrangement enables direct measurement of the pulse where they interact with the sample.