lowautofluorescence

Low autofluorescence refers to the reduced intrinsic emission of light by a sample when it is excited by fluorescence illumination. In fluorescence imaging, autofluorescence from endogenous substances can obscure or confound signals from applied fluorophores, so samples with low autofluorescence are desirable for clearer, more quantitative results. Endogenous fluorophores vary by tissue and organism and include components such as collagen and elastin in connective tissue, lipofuscin in aging cells, NADH and FAD in metabolism, porphyrins from heme pathways, and, in plant tissues, chlorophyll. The spectral profile of autofluorescence depends on excitation wavelength, tissue type, and fixation or mounting conditions.

Measurement and impact are typically assessed by imaging unlabelled samples across multiple channels or using spectral imaging to characterize the autofluorescence signature. Low autofluorescence improves signal-to-noise ratio and can enhance the accuracy of quantitative measurements and colocalization analyses in immunofluorescence and other fluorescent assays.

Strategies to achieve or exploit low autofluorescence include selecting tissues or samples known to exhibit minimal background, choosing excitation and emission windows that avoid strong endogenous fluorescence (for example, moving toward red or near-infrared fluorophores), and applying imaging approaches such as spectral unmixing or lifetime-based separation to distinguish autofluorescence from true signals. Chemical quenching with agents like sodium borohydride or Sudan Black B can reduce certain background signals, and careful sample preparation—such as avoiding fixatives that raise autofluorescence and optimizing mounting media—can help as well. However, quenching may affect fluorophore performance or antigenicity, so controls are essential.

Low autofluorescence is particularly relevant in histology, immunofluorescence, and fluorescence in situ hybridization, where minimizing background enhances image clarity and the reliability of quantitative analyses.