Chromatic shift in multicolour confocal microscopy

Multicolour confocal microscopy has proven to be a successful technique for the analysis of the spatial relationship between different biological structures in the same preparation. However, when the positions of objects are compared, e.g. co‐localization and distance measurements, any positional shift that arises between the colour components is clearly unacceptable. This paper presents a simple technique for measuring with high accuracy the positional shifts that occur between the colour components of an image. Multi‐labelled microbeads were scanned using two or three different detection channels. The position of each microbead was calculated separately for each detection channel. In general, the two calculated positions of the same microbead (one for each channel) are slightly different. This difference is a measure of the positional shift between the colours. This method enables the measurement of shift with a high accuracy (20 nm), and it has been applied to images from several experiments. The results of these experiments will give the reader an impression of typical contributions of different effects (such as chromatic aberration, misalignment of optical components and inaccuracy of the scanning unit) on the amount of positional shift.