Autofluorescence

PaperAutofluorescence

Unmixed Autofluorescence

Label-free Localisation Microscopy SPDM - Super Resolution Microscopy Christoph Cremer

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Autofluorescence is the natural emission of light by biological structures when they have absorbed light or electromagnetic radiation. It is a form of luminescence that is commonly observed in a wide range of biological (such as cells and tissue) and non-biological materials. Autofluorescence is an important phenomenon in the field of biomedical imaging and fluorescence microscopy, as it can be both a tool for studying biological materials and a source of background noise that can interfere with the detection of specific fluorophores (fluorescent markers used to label or highlight biological molecules or structures).

Mechanism

Autofluorescence occurs when molecules known as fluorophores or autofluorescent molecules absorb light at a specific wavelength and then emit light at a longer wavelength. The difference in the absorbed and emitted wavelengths is known as the Stokes shift. Common biological fluorophores include NADH, flavins, aromatic amino acids, and chlorophyll, among others. These molecules can naturally fluoresce when excited by light of the appropriate wavelength, without the need for external fluorescent dyes or labels.

Applications

Autofluorescence has a variety of applications in the field of life sciences and medicine. It is used in fluorescence microscopy to study the structure and function of cells and tissues without the need for external staining. This is particularly useful for live-cell imaging, where the use of non-invasive techniques is crucial. Autofluorescence imaging (AFI) is also employed in clinical settings for the diagnosis and monitoring of diseases. For example, autofluorescence bronchoscopy is used for the early detection of lung cancer by identifying areas of abnormal tissue autofluorescence in the bronchial tubes.

Challenges

One of the main challenges associated with autofluorescence is its potential to interfere with the detection of specific fluorophores used in fluorescence microscopy and other imaging techniques. The background fluorescence from the sample itself can mask the signal from the fluorophores, making it difficult to distinguish between the two. This is particularly problematic when the fluorophore and the autofluorescent molecules have similar excitation and emission spectra. Various strategies, such as the use of spectral unmixing and fluorescence lifetime imaging microscopy (FLIM), have been developed to overcome this issue.

Conclusion

Autofluorescence is a significant phenomenon in the field of biological and medical imaging, offering a non-invasive means of studying biological materials. Despite its challenges, ongoing advancements in imaging technology and techniques continue to enhance its utility and overcome limitations associated with background fluorescence.

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