Eliminating Registration Shifts in Multiple-Exposure Fluorescence Microscopy

When capturing multiple-exposure images or sequential electronic images of specimens stained with different fluorochromes, unavoidable registration shifts often occur due to changes in filter cubes in a standard microscope. These shifts arise from factors such as wedge variation in emission filters and beamsplitters, differences in thickness and alignment of the beamsplitter, and even mechanical vibration from switching cubes. While some applications can tolerate minor shifts, others require more advanced solutions.

Table below outlines several available methods that help mitigate registration errors in applications using multiple probes. These methods include multi-band filter sets, as shown in the figure below, as well as configurations that combine multi-band and single-band filter components to improve registration accuracy. Method 2, for instance, allows for simultaneous visual observation of up to three colors, while methods 3 through 6 can achieve similar multi-color viewing with additional multi-band filters.

This list serves as a guide, showcasing key methods to address registration shifts, although it is not exhaustive and can be adapted for various configurations and applications.