Backward second harmonic generation from starch for in situ, real time pulse characterization in multiphoton microscopy

It is well known that the efficiency and selectivity of 1wo-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 in situ, 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.