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Multiwavelength two-photon excitation

Hands-free operative tunable sources gave in the last years to biologists an user friendly tool to access different regions of the excitation spectra of different multiphoton fluorophores. Therefore, structural and functional imaging, highlighting different structures labeled with flurophores with no overlapping multiphoton excitation cross sections, became easier to be performed. Moreover, novel continuum sources, based on different technologies, suitable to deliver ultrfast pulses in a broad range have then be developed and commercialised, in order to excite multi-photon fluorescence in different regions at the very same time. Usually one has to face trade-off between spectral power density and intensity noise, which obviuosly play a fundamental role in the SNR of the acquired data. Furthermore, a big part of optical power can be wasted into regions of the spectrum which will be never used for multiphoton imaging, and selection with optical filters has to be performed to send on the sample just the single band of interest.

Principle of operation

Our CIrCuS - Controllable Infrared Continuum Source1, suitable for multiphoton imaging, is based on non linear broadening in Photonics Crystal Fibers (PCFs) of femtosecond laser pulses delivered by a Ti:Sa laser tuned around 800nm. In a compact closed setup we also implemented an automated control, based on a patent pending technique, for arbitary amplitude spectral modulation of the continuum light, giving the possibility to select different bands to be delivered to the sample at the same time

Benefits of an unusual approach

In most experiments, the PCFs are pumped in anomalous dispersion region, to obtain the maximum broadening. Instead, we choose to pump mainly in the normal dispersion region, i.e. at wavelengths shorter than Zero Dispersion Wavelength (ZDW), in order to limit the non-linear interaction and favour a greater power density and a lower level of intensity noise, with respect to a broad spectrum. Moreover, pumping in normal region should give flatter spectra and should avoid the soliton fission of the pulse. Furthermore, ZDW sets an upper2 limit in wavelength to the broadening processes.

Everything inside a box

CIrCuS is a one-box system. External knobs provide fine adjustement for fiber injection. Simple software for automated control is included, thus providing the user with everything he/she needs to perform his/her experiments. Single spatial mode output beam (diameter selectable at the order) can be easily coupled to the scanning head of your fluorescence microscope.

References

1 de Mauro et al. Controllable infrared continuum source for multiphoton imaging. Multiphoton Microscopy in the Biomedical Sciences X. Edited by Periasamy, Ammasi; So, Peter T. C.; König, Karsten. Proceedings of the SPIE, Volume 7569, pp. 756919-756919-6 (2010).
2 J. M. Dudley, G. Genty, and S. Coen, “Supercontinuum generation in photonic crystal fiber,” Rev. Mod. Phys. 78, pp. 1135–1184, (2006).


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