Crosstalk | Crosstalk

Key Words: Fluorescence, fluorophore, spectral imaging, FLIM, filter block

Definition:Crosstalk in fluorescence imaging occurs when the excitation and / or emission spectra of two or more fluorophores (and /or autofluorescence) in a specimen overlap making it difficult to isolate the activity of one fluorophore alone

TECHNOLOGY:

Crosstalk can occur during both excitation and emission of different fluorescent proteins and is usually observed by the emission of one fluorophore being detected through the photomultiplier channel or filter combination of another fluorophore. This can be a problem in image interpretation, for example, in co-localization studies and/or quantitative studies. Crossover in excitation tends to occur increasingly towards shorter wavelengths while crosstalk in emission is skewed towards longer wavelengths. Multicolor imaging of fluorescent proteins might, therefore, proceed with the longest wavelength emission probe imaged first, using excitation wavelengths that do not crossover to shorter wavelength probes.

APPLICATIONS:

Multiprobe fluorescence imaging is a key technique in live cell protein trafficking, interaction and localization studies. Crosstalk may present a problem in both widefield and confocal studies.

MICROSCOPE CONFIGURATION:

The problem of cross talk may be overcome by:

• choosing only probes with widely separated excitation and emission profiles
• by using narrow bandwidth filters (although this may also restrict signal)
• by using the fluorescence lifetime to distinguish overlapping probes (rather than emission)
• by using spectral imaging techniques (in combination with unmixing algorithms).

RECOMMENDED SYSTEM:

Nikon's C1si spectral imaging confocal laser microscope system can acquire 32 channels of fluorescence spectra over a 320 nm wide wavelength range in a single pass. By mathematically processing the spectral data of closely overlapping probes (such as GFP, YFP, FITC, and Alexa 488) the C1si cleanly separates emissions from each to yield clear images with no cross-talk. Spectral separation of probe signals from autofluorescence is also possible. Easy switching between the spectral detector and a standard fluorescence detector makes the C1si useful for a wide range of applications.

LINKS:

Imaging parameters for fluorescent proteins

Nikon Note 1: Fluorescence lifetime imaging (FLIM) on a spectral confocal microscope: The ultimate tool for fluorescence resonance energy transfer (FRET) analysis