4Pi microscopy of quantum dot-labeled cellular structures

The most prominent restrictions of fluorescence microscopy are the limited resolution and the finite signal. Established conventional, confocal, and multiphoton microscopes resolve at best ∼200 nm in the focal plane and only ⩾500 nm in depth. Additionally, organic fluorophores and fluorescent proteins are bleached after 104–105 excitation cycles. To overcome these restrictions, we synergistically combine the 3- to 7-fold improved axial resolution of 4Pi microscopy with the greatly enhanced photostability of semiconductor quantum dots. Co-localization studies of immunolabeled microtubules and mitochondria demonstrate the feasibility of this approach for routine biological measurements. In particular, we visualize the three-dimensional entanglement of the two networks with unprecedented detail.