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| Widefield Multichannel Unmixing |
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When the Carl Zeiss LSM 510 Meta launched in 2001 it was the first confocal laser scanning microscope to permit the precise separation of fluorescence signals. In 2003 Multichannel unmixing of regular confocal channels was added with the LSM Software Release 3.2 Add-On. In a logical next step Carl Zeiss now offers "Widefield Multichannel Unmixing", a new module for optimizing conventional multichannel fluorescence images by the precise correction of emission crosstalk between fluorescent channels.
Crosstalk is a phenomenon occurring whenever fluorescent dyes are excited by the excitation light transmitted by more than one filter combination. The problem is serious where the fluorochromes used have greatly overlapping excitation spectra, as for example in case of the spectral variants of fluorescent proteins (BFP, CFP, GFP and YFP). Crosstalk between such dyes often cannot be fully excluded with conventional reflector filter combinations. The result is that a certain percentage of the signal in a fluorescent channel originates from another dye, the emission light of which mixes with the emission signal of the dye anticipated for that channel.
The new module allows crosstalk to be computed from measurements of suitable reference specimens containing only one dye each or by means of the Automatic Component Extraction (ACE) from Carl Zeiss. This improves the applicability of combinations of widely overlapping dyes such as CFP and GFP or YFP and DsRed within a specimen dramatically.
The Widefield Multichannel Unmixing module at a glance
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Coexpression of CFP (blue) and GFP (yellow) in HeLa cells. The illustration shows a 2-channel image before and after unmixing by the Widefield Multichannel Unmixing module.
A comparison is possible by means of the image comparison function:
Left: before unmixing.
Center: unmixed with ACE.
Right: unmixed by crosstalk computation using reference specimens.
Specimen: Dr. Horst Wolff, GSF, Neuherberg, Germany. |
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