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Improving Fluorescence-Based Assay Performance Using Automated Digital MicroscopyDownload
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September 02, 2014
Fluorescence remains one of the most popular detection modes used in cell-based assays primarily due to the wide array of fluorophores available. These can be grouped into three main families of fluorophore:
- Reactive dyes that can be used to make fluorescent conjugates of biomolecules (i.e. antibodies) that can be used to probe cellular processes
- Fluorogenic probes that upon binding undergo significant changes in fluorescence quantum yield
- Photoproteins like the family of GFP variants that can be transfected into cells as reporter systems or fusion proteins.
One of the main limitations of fluorescence detection is background fluorescence, either from autofluorescence from endogenous cellular material, impurities from reagents added to the assay, unbound fluorescent conjugates or fluorogenic probes, or unreacted photoprotein. Autofluorescence and background from impurities can be reduced by the use of red-shifted fluorophores; unbound fluorescent conjugates can be removed through gentle wash cycles that allow cells to remain adhered to plasticware; but for homogeneous assays in microplates that rely on simple mix and read workflows, background from unbound probe or unreacted photoprotein can be a problem that limits assay performance.
Here we demonstrate three unique ways in which the incorporation of imaging, in addition to cellular analysis, allow for a more accurate analysis of the event taking place within the microplate well, and improve assay performance.