4Pi microscopy with linear fluorescence excitation.
Practical 4Pi microscopy has so far exclusively relied on multiphoton excitation of fluorescence, because the nonlinear suppression of contributions from higher-order sidelobes was mandatory for unambiguous axial superresolution. We show that novel lenses of 74 degrees semiaperture angle enable biological 4Pi microscopy with regular one-photon fluorescence excitation, thus increasing the signal and reducing system complexity and cost. An axial resolution of 95 nm, corresponding to a more than fourfold improvement over confocal microscopy, is verified in the imaging of microtubules in mammalian cells.
Lang
Marion C
MC
High Resolution Optical Microscopy Division, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.
Engelhardt
Johann
J
Hell
Stefan W
SW
D003198:Computer Simulation; D007089:Image Enhancement / Q000295:instrumentation* / Q000379:methods*; D007090:Image Interpretation, Computer-Assisted / Q000379:methods*; D007909:Lenses; D016014:Linear Models; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation* / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity