What is Background Fluorescence?
Background fluorescence refers to the unwanted fluorescent signals that are not specific to the target structures in a histological sample. This phenomenon can obscure or interfere with the specific signals, making it challenging to obtain accurate and clear images. It is often a significant issue in
immunofluorescence and other fluorescence-based imaging techniques.
Causes of Background Fluorescence
Several factors can contribute to background fluorescence in histological samples: Autofluorescence: This is the natural emission of light by biological structures when they absorb light. Common sources include collagen, elastin, and certain cellular metabolites such as NADH and flavins.
Non-specific binding: Fluorescent dyes or antibodies may bind non-specifically to tissue components, generating unwanted signals.
Fixation: Some fixation methods, like formaldehyde fixation, can increase autofluorescence due to chemical modifications of tissue components.
Mounting media: Some mounting media have intrinsic fluorescence, which can add to the background signal.
Incomplete washing: Residual unbound fluorophores can remain in the tissue, contributing to background fluorescence.
Blocking agents: Using blocking agents like BSA, serum, or commercial blocking buffers can reduce non-specific binding of antibodies.
Optimize antibody concentration: Using the correct concentration of primary and secondary antibodies can reduce non-specific signals.
Quenching agents: Certain reagents can be used to quench autofluorescence. Sodium borohydride, for example, can reduce aldehyde-induced autofluorescence.
Choose the right fluorophore: Selecting fluorophores with emission spectra distinct from the autofluorescent background can improve signal-to-noise ratio.
Proper washing: Ensure thorough washing steps to remove unbound fluorescent probes.
Accuracy: High background fluorescence can lead to misinterpretation of results, affecting the accuracy of qualitative and quantitative analyses.
Signal-to-noise ratio: Reducing background fluorescence improves the signal-to-noise ratio, enabling clearer and more precise detection of target signals.
Reproducibility: Minimizing background fluorescence enhances the reproducibility of experiments, which is essential for scientific validity.
Examples of Techniques to Assess and Control Background Fluorescence
Several techniques are employed to assess and control background fluorescence: Control samples: Using negative controls (samples without primary antibody) can help identify the level of background fluorescence.
Spectral unmixing: This computational technique separates overlapping fluorescence signals based on their spectral properties.
Confocal microscopy: This imaging technique improves contrast by eliminating out-of-focus light, which can reduce background fluorescence.
Multiphoton microscopy: This advanced technique uses longer wavelength light, which can penetrate deeper into tissues and reduce autofluorescence from superficial layers.
Conclusion
Background fluorescence remains a significant challenge in fluorescence-based histological techniques. Understanding its causes and employing strategies to minimize it can greatly improve the quality and accuracy of histological analyses. By addressing background fluorescence, researchers can obtain clearer images, leading to more reliable and reproducible scientific findings.
