Probes for Two-Photon-Activation FPALM

Abstract

Biplane fluorescence photoactivation localization microscopy (FPALM) [1], a localization based super-resolution technique, uses photo-activatable fluorescent proteins (PAFPs) to image samples at resolutions beyond the diffraction limit. These PAFPs undergo a controlled change in their emission spectrum upon absorption of a photon typically of the UV range. By repeatedly activating and localizing PAFPs in the sample a three-dimensional map of molecule positions is generated that has a far better resolution than typical light microscopy techniques can achieve.For improved performance, especially when imaging thicker samples, the 405 nm activation light can be replaced by a focused mode-locked laser beam of 700-1000 nm wavelength. This type of setup then uses two-photon-activation to confine the activation axially to the focal plane [2-5], increasing the fraction of usable labels in the sample because molecules out of focus will not be activated and bleached. For the same reason background will be reduced which opens up the way to imaging thicker samples that exhibit too high background levels in conventional FPALM.In order to choose the best probe, it is important to know how each one performs using two-photon-activation. Here we present a method that allows determining the two-photon-activation cross section of any photo-activatable fluorescent probe. We show results for a number of commonly used established and new PAFPs as well as images recorded using two photon activation.[1] M.F. Juette, et al., Nat. Methods 5(6):527-9 (2008)[2] M Schneider, et al., Biophys J. 89(2):1346-52 (2005)[3] S. Ivanchenko, et al., Biophys J. 92(12):4451-7 (2007)[4] J Folling, et al., Chemphyschem 9(2):321-6 (2008)[5] A.G. York, et al., Nat. Methods 8(4):327-33 (2011)