Signal enhancement in multiphoton TIRF microscopy by shaping of broadband femtosecond pulses.

We demonstrate that pulse shaping of a broadband Ti:sapphire laser can result in almost an order of magnitude increase in the sensitivity and signal to background ratio (SBR) of multiphoton total internal reflection fluorescence (TIRF) microscopy. We produced transform-limited pulses of 15 fs duration at the sample, and observed a 8-fold enhancement in the fluorescence of CdSe/ZnS quantum dots via two-photon objective-type TIRF excitation. There was a concomitant 6-fold increase of the SBR upon compression of the pulse duration. Enhancement of non-linear evanescent imaging has recently been demonstrated using surface-plasmons [Opt. Express 17, 5987 (2009)] and structured substrates [Opt. Express 18, 23218 (2010)]. Our approach of ultrafast pulse shaping could be used alone or combined with these new methods to offer significant gains in image quality.