Clean localization super resolution microscopy for 3D biological imaging
We propose clean localization microscopy (a variant of fPALM) using a molecule filtering technique. Localization imaging involves acquiring a large number of images containing single molecule signatures followed by one to one mapping to render a super resolution image. In principle, this process can be repeated for other z planes to construct a 3Dimage. But, single molecules observed from off focal planes result in false representation of their presence in the focal plane, resulting in incorrect quantification and analysis. We overcome this with a single molecule filtering technique that imposes constraints on the diffraction limited spot size of single molecules in the image plane. Calibration with sub diffraction size beads puts a natural cutoff on the actual diffraction limited size of single molecules in the focal plane. This helps in distinguishing beads present in the focal plane from those in the off focal planes thereby providing an estimate of the single molecules in the focal plane. We study the distribution of actin (labeled with a photoactivatable CAGE 552 dye) in NIH 3T3 mouse fibroblast cells.