Fluorescence Intensity Measurements
The main problem we cannot escape when making fluorescence measurements is bleaching of the fluorochrome. Bleaching is likely to be at least as significant a factor as any properties of the microscope and measurement system. Second to this is the problem of attenuation with depth in the sample, which is very hard to quantify because it is a mixture of absorption, scattering, and spherical aberration and depends strongly on local properties of the sample. Many problems are best solved by using measures that are independent of intensity, such as ratios (later in this chapter) or fluorescence lifetime (Chapter 15), but for many measurements — such as our example of the DNA content of a nucleus —only intensity measurement will give us the answer.