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- Fluorescent dyes serve as markers for labeling specific structures or proteins in a cell or tissue.
- With several proteins labeled, different cellular structures can be studied simultaneously.
- Cells and tissues can express a number of different fluorescent proteins.
Requirements: The identification and visualization of the fluorescent signals must be unambiguous and reproducible. The exciting light needs to be efficiently separated from the emitted fluorescence.
Capabilities of Carl Zeiss systems
With standard markers, separation between excitation and emission light is achieved by means of Multitracking.
This can be assisted by the MultiChannel Unmixing function.
Where emission signals greatly overlap, signal separation is achieved by means of Emission Fingerprinting. In case of unknown or missing emission spectra, Automatic Component Extraction can identify the spectra. With Online Fingerprinting this procedure directly yields the result – unambiguously separated fluorescence signals.
In multiphoton microscopy, the Excitation Fingerprinting function makes it possible to use excitation spectra for signal separation.
Multichannel imaging
In multifluorescence imaging, different fluorochromes are used to target specific areas in the specimen. The goal is to image each fluorochrome in individual detection channels. Band pass detection is appropriate to separate the fluorescent signals into the individual detection channels if only minor spectral overlap of the emission and the excitation spectra is present.
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| Excitation and emission signal as a function of wavelength for two spectrally separated fluorochromes (schematic) |
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Multitracking
In most applications, fluorochromes have overlapping emission spectra. Hence, the emission signals can not be separated completely into different detection channels resulting in crosstalk. However, if the fluorochromes have distinct excitation spectra, the fluorochromes can be excited sequentially using one excitation wavelength at a time.
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| Excitation and emission signal as a function of wavelength for two fluorochromes that have overlapping emission spectra (schematic). |
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Emission Fingerprinting
lf the excitation and the emission spectra are strongly overlapping, the fluorochromes are excited simultaneously with a single excitation wavelength. Dichroic filters are no longer suitable for separating the fluorescent signals into individual detection channels. lnstead the whole spectral signature is used to separate the fluorochromes.
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| Excitation and emission signal as a function of wavelength for two fluorochromes that have strongly overlapping emission and excitation spectra (schematic). |
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Excitation Fingerprinting
Besides the spectral signature of the emission signal of a dye one can also use the changes in intensity of a signal, which occur at different excitation wavelengths, for separation. Therefore this method, which can be used in Multiphoton Microscopy, allows to identify overlapping emission signals from deep tissue layers.
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