Two-color intranuclear distance measurements of gene regions in human lymphocytes

摘要

The method of Spectral Precision Distance Microscopy (SPDM) has been used to determine distances between two FISH (Fluorescence-in situ-Hybridization)-labeled gene regions on chromosome 9. To this end we applied methods to correct for chromatic aberrations of the microscope optics alone and also of the sample induced aberrations due to mismatch of the refractive indices. Using a confocal microscope and a threshold based position determination algorithm, positions could be measured with an accuracy of about 65 nm inside of fixed cell nuclei. Distances obtained from the measurements have been verified using a 3D computer model of the cell nucleus. In principle, this SPDM approach could be combined with novel fluorescence microscopes to obtain structural information well below the optical resolution. At present the precision limit of the distance measurements is set by variations of the refractive index throughout the specimens.