A three-dimensional (3D) laser-scanning confocal microscope was developed for multicolor 3D co-localization of single fluorescent molecules immobilized at a temperature of 1.5 K. The image quality was tested by taking 3D emission image of single quantum dots with three different excitation wavelengths of 635, 532 and 405 nm. The 3D image size was all within 1.2 times of the diffraction-limited size. From the centroid position of the 3D image taken with 635- and 532-nm excitations, 3D co-localization precision of single dot was determined to be 3 nm in the lateral (x, y) and 18 nm in the axial (z) directions. (C) 2013 Elsevier B.V. All rights reserved.