Abstract
Single molecule localization microscopy enables biological samples to be optically observed with sub-diffraction resolution. Here we report new progress in 3D nanoscopic imaging of cellular and subcellular structures by our newly developed virtual volume photoactivated localization microscopy (VVPALM), in which a tilted mirror is used to generate side view of biological samples in addition to the front view, therefore providing precise single molecule localization in three dimensions. VVPALM brings advantages including high efficiency to use detected photons, minimum vulnerability to optical aberration, and simplicity of implementation. VVPALM is ideally compatible with weak chromophores, such as fluorescent proteins. We have used VVPALM to probe the localization and organization of various bacterial proteins, which are fused with photoactivatable proteins, inside or on the membrane of single Escherichia coli cells with sub-100 nm resolution in 3D. We have also combined VVPALM with label-free PAINT (points accumulation for imaging in nanoscale topography) technique to measure the nanoscale morphology of rat neuronal dendrites. Advances in multi-color nanoscopic imaging for protein 3D colocalization will also be presented.
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