Spectral imaging fluorescent emission spectrum

As mentioned above, spectral imaging microscopy is a form of multidimensional fluorescent microscopy where a fluorescent emission spectrum is acquired at each coordinate location in the sample. This mode of imaging has been implemented for wide field, confocal, and two-photon laser scanning microscopy, and several excellent... 

Dark adapted samples (in a Hepes buffer, pH 7.2) were illuminated for 3 seconds by white light and the emission spectrum of delayed fluorescence was measured at room temperature, 1 second after the illumination (6) by using a recently developed high resolution spectroscopy system based on an imaging photomultiplier tube (PIAS, Hamamatsu Photonics, Japan) featuring 1 nm resolution in the spectral range of approximately 300 nm to 850 nm (5). 

The overlap of different fluorophore spectra due to their wide emission range is an inherent problem of multichannel fluorescence imaging (Fig. IB). In the absence of additional signals, a fluorophore can be detected through a longpass filter that covers its whole emission spectrum. If additional fluorophores are present, a bandpass filter aroimd the emission peak is required to constrict the spectral detection range and to thus reduce the crosstalk. 

Fig. 2 Spectral imaging of fluorescence signals. Contributions of CFP, GFP and YFP to eight successive spectral channels are shown. The distribution of emission signal to the channels is a direct representation of the fluorophore emission spectrum and constitutes a spectral signature. With linear unmixing using these spectral signatures as reference, even combined and mixed signals can be clearly separated into the fluorophores that contribute to the total signal...

For the sake of brevity we will use the acronym FSFD for full-spectrum fluorescence detector. There have been a few reports of FSFDs. There have been a variety of optical elements, including vidicons (television-type cameras with associated storage devices) and diode-arrays. There have been a few publications describing the use of FSFDs, many of which were used in PAH analyses. The PAHs are generally highly fluorescent and their spectra are very rich with many bands in both the excitation and the emission spectra. Two examples of PAH fluorescence spectral pairs are shown in Fig. 21.8 and 21.9. The first shows the common mirror-image pattern found for many PAHs, but the latter shows the asymmetrical one seen for many common highly condensed PAHs. 

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