Temporal Focusing, Line and Point-Scanning Two-Photon Fluorescence Microscopy for In Vivo Imaging: A Comparative Study of Signal Levels and Bleaching Rates

Abstract

Two-photon excitation fluorescence (TPEF) microscopy is a widely used technique that benefits from inherent optical sectioning properties, low scattering and low unwanted absorption from biological samples. Its most common implementation is similar to confocal fluorescence microscopy, consisting of scanning the sample with a tightly focused laser beam to build an image pixel by pixel[1]. Point-scanning techniques have the intrinsic drawback that their temporal resolution is limited, reducing their applicability to dynamic samples. New techniques have emerged that allow faster TPEF imaging by making use of line scanning[2], wide-field temporal focusing[3,4], or a combination of the two[5].Because of the quadratic relationship between two-photon fluorescence signal and excitation intensity, these methods are not equivalent in terms of achievable signal for a given speed, field of view, and excitation laser characteristics. Since photo-bleaching mechanisms occur via pathways involving multiple energy levels, the photo-bleaching rate also depends non-linearly on the excitation intensity[6].We present here a comparative quantitative study of the signal levels and photo-bleaching rates for these different TPEF techniques, using in vivo imaging conditions and widely-used fluorescent proteins. This study will help scientists to choose what technique is best for their particular conditions and requirements.[1] W. Denk, J. Strickler, W. Webb, Science (80-. ). 248 (1990) 73-76.[2] G.J. Brakenhoff, J. Squier, T. Norris, a C. Bliton, M.H. Wade, B. Athey, J. Microsc. 181 (1996) 253-9.[3] D. Oron, E. Tal, Y. Silberberg, Opt. Express 13 (2005) 1468-76.[4] T. Schrodel, R. Prevedel, K. Aumayr, M. Zimmer, A. Vaziri, Nat. Methods (2013).[5] E. Tal, D. Oron, Y. Silberberg, Opt. Lett. 30 (2005) 1686.[6] S. Kalies, K. Kuetemeyer, a Heisterkamp, Biomed. Opt. Express 2 (2011) 805-16.