Autofluorescence Quenching
Autofluorescence Quenching - Related Videos
10:57
Quenching and Boiling
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2023
Source: Alexander S Rattner, Sanjay Adhikari, and Mahdi Nabil; Department of Mechanical and Nuclear Engineering, The Pennsylvania State University, University Park, PA Controlled heating followed by rapid cooling is an important element of many materials processing applications. This heat-treating procedure can increase material hardness, which is important for cutting tools or surfaces in high wear environments. The rapid cooling stage is called quenching, and is often performed by immersing...
07:22
Quantitative Fundus Autofluorescence for the Evaluation of Retinal Diseases
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Cited by 22 •
2016
The retinal pigment epithelium (RPE) supports the sensory retina through recycling visual cycle byproducts, which accumulate as lipofuscin. These products are autofluorescent and can be qualitatively imaged in vivo. Here, we describe a method to quantitatively image RPE lipofuscin using confocal scanning laser ophthalmoscopy.
07:36
Autofluorescence Imaging to Evaluate Cellular Metabolism
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Cited by 15 •
2021
This protocol describes fluorescence imaging and analysis of the endogenous metabolic coenzymes, reduced nicotinamide adenine (phosphate) dinucleotide (NAD(P)H), and oxidized flavin adenine dinucleotide (FAD). Autofluorescence imaging of NAD(P)H and FAD provides a label-free, nondestructive method to assess cellular metabolism.
07:47
Anti-virulent Disruption of Pathogenic Biofilms using Engineered Quorum-quenching Lactonases
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Cited by 7 •
2016
Quorum-quenching enzymes are anti-virulent and anti-bacterial options that can mitigate pathogenesis without risk of incurring resistance, by preventing the expression of virulence factors and genes associated with antibiotic resistance and biofilm formation. In this study, we report a method that demonstrates the efficacy of quorum-quenching enzymes in bacterial biofilm disruption.
09:19
Simultaneous Label-Free Autofluorescence Multi-Harmonic Microscopy
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Cited by 3 •
2025
This protocol presents a step-by-step guide for the Simultaneous Label-free Autofluorescence Multi-harmonic (SLAM) microscopic technique, including details on how to generate the laser light source, prepare a tissue sample, conduct imaging, and analyze the data. SLAM advances nonlinear microscopy by measuring four complementary label-free contrasts to investigate the tissue microenvironment.
FAQs
Its main analytical benefit is improving signal-to-background contrast. Endogenous fluorescence can contribute unwanted emission near the wavelengths used to detect antibody-linked dyes, making specific labeling harder to distinguish. Reducing that background allows fluorescent signals associated with immune-cell or pathogen markers to appear more clearly, supporting more reliable microscopy and interpretation of complex biological specimens.
Tissue pigments and extracellular matrix components are important sources of endogenous fluorescence identified in the provided context. Their emission can overlap with the spectral range used for labeled antibodies, adding background to immunofluorescence images. Quenching these contributions helps separate marker-associated fluorescence from signal produced by the specimen itself.
Quenching reagents or treatments are selected and applied to reduce emission from endogenous molecules while preserving fluorescence from antibody-linked dyes. This distinction matters because the goal is not simply to make the specimen less fluorescent. It is to preferentially improve visibility of the specific signal used to identify immune or pathogen-associated markers.
Spectral overlap can cause natural tissue emission and antibody-linked fluorescence to appear in similar detection channels. When that occurs, background may obscure or complicate the interpretation of specific markers. Reducing endogenous emission makes the remaining signal easier to distinguish, which is particularly valuable when immunofluorescence is performed on biologically complex specimens.
The approach is incorporated by applying a quenching reagent or treatment to the biological specimen as part of immunofluorescence preparation, with the aim of reducing endogenous emission before or during signal assessment. The treated sample is then examined for antibody-linked fluorescence. The relevant outcome is clearer separation between specific labeling and specimen-derived background.
It is especially useful when tissue or cellular samples contain intrinsic fluorescence that interferes with detection of immune-cell markers or pathogen-associated signals. Such interference can occur in complex immunological and infectious disease specimens. Lowering the background helps investigators distinguish biologically relevant fluorescence and interpret marker localization or presence more confidently.
Reduced autofluorescence can produce clearer microscopy images and stronger signal-to-background contrast. These improvements make specific fluorescent markers easier to distinguish from surrounding tissue-derived emission. They also support more reliable image analysis, because measurements and visual interpretations are less confounded by natural fluorescence from pigments or extracellular matrix components.
In infectious disease specimens, background control helps separate fluorescence linked to pathogen-associated markers from emission generated by the surrounding biological material. This distinction can improve the visual interpretation of labeled targets in complex samples. By making specific signals easier to resolve, the method strengthens microscopy-based assessment of immune and infection-related features.