Reconstruction of axial tomographic high resolution data from confocal fluorescence microscopy: a method for improving 3D FISH images

Fluorescent confocal laser scanning microscopy allows an improved imaging o f microscopic objects in three dimensions. However, the resolution along th e axial direction is three times worse than the resolution in lateral direc tions. A method to overcome this axial limitation is tilting the object und er the microscope, in a way that the direction of the optical axis points i nto different directions relative to the sample. A new technique for a simu ltaneous reconstruction from a number of such axial tomographic confocal da ta sets was developed and used for high resolution reconstruction of 3D-dat a both from experimental and virtual microscopic data sets. The reconstruct ed images have a highly improved 3D resolution, which is comparable to the lateral resolution of a single deconvolved data set. Axial tomographic imag ing in combination with simultaneous data reconstruction also opens the pos sibility for a more precise quantification of 3D data. The color images of this publication can be accessed from http://www.esacp. org/acp/2000/20-1/heintzmann.htm. At this web address an interactive 3D vie wer is additionally provided for browsing the 3D data. This java applet dis plays three orthogonal slices of the data set which are dynamically updated by user mouse clicks or keystrokes.