Abstract
Bio-imaging is a key technique in tracking and monitoring important biological processes and fundamental biomolecular interactions, however the interference of background autofluorescence with targeted fluorophores is problematic for many bio-imaging applications. This study reports on two novel methods for reducing interference with cellular autofluorescence for bio-imaging. The first method uses fluorescent nanodiamonds (FNDs), containing nitrogen vacancy centers. FNDs emit at near-infrared wavelengths typically higher than most cellular autofluorescence; and when appropriately functionalized, can be used for background-free imaging of targeted biomolecules. The second method uses europium-chelating tags with long fluorescence lifetimes. These europium-chelating tags enhance background-free imaging due to the short fluorescent lifetimes of cellular autofluorescence. In this study, we used both methods to target E-selectin, a transmembrane glycoprotein that is activated by inflammation, to demonstrate background-free fluorescent staining in fixed endothelial cells. Our findings indicate that both FND and Europium based staining can improve fluorescent bio-imaging capabilities by reducing competition with cellular autofluorescence. 30 nm nanodiamonds coated with the E-selectin antibody was found to enable the most sensitive detective of E-selectin in inflamed cells, with a 40-fold increase in intensity detected.
Highlights
Background autofluorescence is a major issue for the bio-imaging of cells and tissues
Our results demonstrated that both ligand conjugated fluorescent nanodiamonds (FNDs) and SA-BHHTEGST-Eu can enable the background-free detection of E-selectin within highly autofluorescent mouse brain endothelial cells
We have demonstrated two different probe based approaches for reducing cellular background autofluorescence when imaging highly autofluorescent brain endothelial cells
Summary
Background autofluorescence is a major issue for the bio-imaging of cells and tissues. Differentiating between cellular autofluorescence and fluorescent staining from a dye is not always possible, in cases where the expression of targeted molecules is low and the dye’s staining is weak In such cases, only major changes in fluorescent intensity can be detected due to background autofluorescence. The FNDs are not susceptible to photobleaching like conventional fluorophores[4], nor do they exhibit photo-blinking such as observed with quantum dots[7] These properties are the major advantage for the use of FNDs for bio-imaging, in particular for long-term tracking studies[8]. While the core of the FND particles is stable, their surface is reactive This allows for easy bio-functionalization and specific targeting of an analyte within a biological environment[10]. This versatile ‘FND-PEG-SA’ scaffold can bind to any cellular target for which a biotinylated ligand can be generated
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