What kind of microscope do I need for ratiometric dyes?

Fluorescence ratio imaging is a spectroscopic technique and is usable for any technique that can be expressed by the relationship of signals at two or more wavelengths. You’ll need to use an epifluorescence microscope for ratiometric dyes. This is a specific type of fluorescence microscope in which both the illuminated light and the emitted light travel through the same objective lens. The name is derived from the Greek term ‘epi’, which means ‘same’.

In epifluorescence microscopy, the amount of illumination is maximized by passing a parallel beam of light from a high-intensity light source directly upward through the sample. Epifluorescence microscopy is particularly useful when imaging thick samples that are more than10µm deep as it allows the illumination beam to penetrate the full depth of the sample.