What is autofluorescence in flow cytometry?

MIT Flow Cytometry Core Facility. Page 2. Autofluorescence. Cells contain molecules, which become fluorescent when excited by UV/Visual radiation of suitable wavelength. This fluorescence emission, arising from endogenous fluorophores, is an intrinsic property of cells and is called auto-fluorescence.

What causes tissue autofluorescence?

Tissue autofluorescence has been attributed to many factors including endogenous tissue elements such as collagen, tissue processing techniques, particularly formalin fixation, and reagents such as serum that are applied to tissues during immunofluorescence protocols (Baschong et al.

What is autofluorescence in microscopy?

Autofluorescence is the natural emission of light by biological structures such as mitochondria and lysosomes when they have absorbed light, and is used to distinguish the light originating from artificially added fluorescent markers (fluorophores).

What is the function of autofluorescence?

In biology, autofluorescence is often regarded as a nuisance, masking the specific fluorescence of introduced substances, but it usually provides a guide to the morphology of the tissue, and is also worthy of study in its own right. In geology, fluorescence microscopy is widely used to study coal.

Are dead cells autofluorescence?

Dead cells tend to be more autofluorescent than live cells, bind antibody non-specifically, and are difficult to completely eliminate from analysis based solely on forward and side scatter.

What is bacterial autofluorescence?

Autofluorescence is the spontaneous fluorescence of materials without added dyes. Many people are responsible for this work but principal credit goes to Cindy Maddera, who just received her Master’s in my lab. Also contributing were Shelly Theiss, and Amy Huseby.

How do I fix autofluorescence?

Use fluorophores that emit in a wavelength further from the autofluorescence compounds in your sample. Typically, far-red wavelength fluorophores such as CoralLite 647 are best for this. Commercially available reagents such as TrueVIEW (VectorLabs), have been shown to reduce autofluorescence from multiple causes.

What is tissue autofluorescence?

Autofluorescence is a general term describing the background fluorescence in tissue sections unrelated to the specific signal generated during an IF assay. Tissue components such as red blood cells (RBCs) and collagen are strongly fluorescent, making it difficult to discern between relevant signal and background.

How do I stop autofluorescence?

5 Tips to Reduce Autofluorescence

  1. Use a lower concentration of FCS in the staining buffer.
  2. Remove dead cells & other debris.
  3. Lyse RBCs properly & remove lysed contents.
  4. Lower PFA concentration & avoid storing cells in PFA for long durations.
  5. Make the right fluorochrome choices.

What causes autofluorescence in bacteria?

All prokaryotic and eukaryotic cells exhibit an intrinsic natural fluorescence (autofluorescence; AF) due to the presence of different fluorescent cellular structural components and metabolites, such as flavins, nicotinamide-adenine dinucleotide (NAD), aromatic amino acids, lipofuscins, advanced glycation end products.