Some structures, biological organisms, and general microscopy samples can naturally exhibit fluorescence, known as autofluorescence. This is different from fluorescence from labeled samples, but it often shares similar wavelengths, meaning that autofluorescence microscopy samples can obscure artificially added fluorescence and interferes with detection, reducing signal. It is important to know if your samples exhibit autofluorescence, as this will affect any fluorescence imaging done unless specific wavelengths are used to avoid it.

Common examples of autofluorescent objects are mitochondria, lysosomes, collagen and some amino acids such as tryptophan, tyrosine, and phenylalanine. Most notably, autofluorescence is common in plants due to their use of chlorophyll and other fluorescent molecules like lignins and carotenes. Fig.2 shows the different colors of autofluorescence from an unlabeled Scots pine sample.