3D-Resolution In FPALM/PALM/STORM

Abstract

Light microscopy is one of the most widespread imaging techniques used by biologists, but even the best traditional microscopes are limited by diffraction to a resolution of about 250nm, leaving many sub-cellular structures in an unresolved blur. Over the last few years, several groups have pioneered localization techniques to surpass the resolution limit1-4; however those techniques were limited to two dimensions. Recently, two techniques have been employed that extend localization into the third, axial dimension5,6. Biplane fluorescent photoactivatable localization microscopy6 (BP-FPALM) splits the detected fluorescence into two paths7, one slightly longer than the other to image two planes at different depths in the sample. The other method introduces a cylindrical lens to the detection path8, which causes astigmatism in the detected fluorescence5. This results in a stretch along one of the two lateral axes depending on the axial position of the fluorescent particle. This work determines the best optical parameters for each method in order to localize over the largest axial range with best possible uniformity in localization accuracy.1. Hess, S. T. et al., Biophys. J. 91, 4258 (2006).2. Betzig, E. et al., Science 313 (5793), 1642 (2006).3. Rust, M. J. et al., Nat. Methods 3 (10), 793 (2006).4. Egner, A. et al., Biophys. J. 93 (9), 3285 (2007).5. Huang, B. et al., Science 319 (5864), 810 (2008).6. Juette, M. F. et al., Nat Methods 5 (6), 527 (2008).7. Berg, H. C., Rev Sci Instrum 42 (6), 868 (1971).8. Kao, H. P. et al., Biophys J 67 (3), 1291 (1994).