ABSTRACT
A model based method for the accurate quantification of the 3D structure of fluorescently labelled cellular objects similar in size to the optical resolution limit is presented. This method is applied to both simulated confocal images of chromatin structures and to real confocal data obtained on a Fluorescence in situ Hybridization (FISH) labelled gene domain. The model assumes that the object is composed of a small number of discrete points which are convolved with the microscope point spread function to give the image. Fitting this model to image data results in a method to assess object structure which is accurate, shows a low bias, and does not require user intervention or the potentially subjective setting of a threshold.
Subject(s)
Chromatin/ultrastructure , Computer Simulation , Models, Biological , snRNP Core Proteins , Cell Nucleus/ultrastructure , DNA Probes , Fluorescent Dyes , Genes , Humans , In Situ Hybridization, Fluorescence/methods , Lymphocytes/ultrastructure , Microscopy, Confocal/methods , Molecular Conformation , Prader-Willi Syndrome/genetics , Sensitivity and Specificity , snRNP Core Proteins/genetics , snRNP Core Proteins/ultrastructureABSTRACT
We present a novel technique of far-field localization nanoscopy combining spectral precision distance microscopy with widely used fluorochromes like the Green Fluorescent Protein (GFP) derivatives eGFP, EmGFP, Yellow Fluorescent Protein (YFP) and eYFP, synthetic dyes like Alexa 488 and Alexa 568, as well as fluoresceine derivates. Spectral precision distance microscopy allows the surpassing of conventional resolution limits in fluorescence far-field microscopy by precise object localization after the optical isolation of single signals in time. Based on the principles of this technique, our novel nanoscopic method was realized for laser optical precision localization and image reconstruction with highly enhanced optical resolution in intact cells. This allows for spatial assignment of individual fluorescent molecules with nanometre precision. The technique is based on excitation intensity dependent reversible photobleaching of the molecules used combined with fast time sequential imaging under appropriate focusing conditions. A meaningful advantage of the technique is the simple applicability as a universal tool for imaging and investigations to the major part of already available preparations according to standard protocols. Using the above mentioned fluorophores, the positions of single molecules within cellular structures were determined by visible light with an estimated localization precision down to 3 nm; hence distances in the range of 10-30 nm were resolved between individual fluorescent molecules allowing to apply different quantitative structure analysis tools.
