Partial internal reflections on total internal reflection fluorescent microscopy

Microscopy, especially fluorescence microscopy, has proven to be a powerful method for studying biological processes. Unfortunately, some of the same features that make biological membranes powerful (for example, all of the action taking place across a narrow 4nm film) also make it difficult to visualize by fluorescence. Over the past 30 years, numerous tricks have been developed to narrow the plane over which data is collected. One approach, total internal reflection (TIR) fluorescence microscopy, is particularly well suited for studying membrane events. A key issue to address when using TIR to tackle a new biological problem is: how can one judge whether the signals being observed are actually the biological phenomena that one wishes to study?