Since the introduction of two-photon microscopy by Webb and Denk it has been known that the two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG) signal intensity should be linearly proportional to the inverse of the pulse duration. To utilize ultrashort pulses, however, chromatic dispersion must be compensated. We use multiphoton intrapulse interference phase scan (MIIPS®) method [1] to measure and then cancel phase distortions, obtaining transform limited (TL) pulses at the focal plane.

TPEF/SHG imaging with TL pulses and laser pulses compensated for group delayed dispersion (GDD) on: (a) SAOS-2 fixed cells stained with phalloidin 568. TPEF signal obtained with TL pulses had an 11-fold greater intensity compared to the signal acquired when GDD-only compensation was used. (b) U2OS living cell stained with MitoTracker 488. The measured gain in TPEF signal intensity was ~6. (c) Mouse liver tissue cross-section stained with MitoTracker 488 and phalloidin 568. The gain factor was ~7. (d) SHG image of a fresh rat tendon with the observed gain of ~19. The images were taken sequentially starting with GDD-only using a Zeiss LD C-APOCHROMAT 40x/1.1 NA objective. Image size is 150 µm. TL pulse duration for all images is 12 to 13 fs. Image courtesy of Dantus Research Group.

Reference: Y. Coello, V. V. Lozovoy, T. C. Gunaratne, B. Xu, I. Borukhovich, C. -h. Tseng, T. Weinacht, and M. Dantus, "Interference without an interferometer: a different approach to measuring, compressing, and shaping ultrashort laser pulses," J. Opt. Soc. Am. B 25, A140-A150 (2008)

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