What is the best way to reduce “bleedthrough” when fluorescein and rhodamine are used simultaneously? A.Using a stronger excitation for rhodamine. B.Using an optimal combination of fluorescence filters. C.Using less rhodamine than fluorescein. D.By imaging each fluorescent molecule separately and waiting several seconds between
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What is the best way to reduce “bleedthrough” when fluorescein and rhodamine are used simultaneously? |
- A.Using a stronger excitation for rhodamine.
- B.Using an optimal combination of fluorescence filters.
- C.Using less rhodamine than fluorescein.
- D.By imaging each fluorescent molecule separately and waiting several seconds between each image, to ensure that all fluorescence has been emitted.
Due to the extremely wide bandwidths and asymmetrical spectral profiles displayed by many common fluorophores, bleed-through (often referred to as crossover or crosstalk) of fluorescence emission is a fundamental issue that needs to be addressed in both widefield and laser scanning confocal fluorescence microscopy.
The phenomenon often manifests as a single fluorophore's emission being picked up by a photomultiplier or a filter set designated for another fluorophore.
Bleed-through artefacts frequently make it more difficult to interpret the results of experiments, especially when fluorophores are colocalized within cells or when precise measurements are required, as in studies of resonance energy transfer (FRET) and photobleaching (FRAP).
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