Spectral imaging and its applications in live cell microscopy.
In biological microscopy, the ever expanding range of applications requires quantitative approaches that analyze several distinct fluorescent molecules at the same time in the same sample. However, the spectral properties of the fluorescent proteins and dyes presently available set an upper limit to the number of molecules that can be detected simultaneously with common microscopy methods. Spectral imaging and linear unmixing extends the possibilities to discriminate distinct fluorophores with highly overlapping emission spectra and thus the possibilities of multicolor imaging. This method also offers advantages for fast multicolor time-lapse microscopy and fluorescence resonance energy transfer measurements in living samples. Here we discuss recent progress on the technical implementation of the method, its limitations and applications to the imaging of biological samples.
Zimmermann
Timo
T
Advanced Light Microscopy Facility and Cell Biology/Cell Biophysics Programme, European Molecular Biology Laboratory, Meyerhofstr. 1, D-69117 Heidelberg, Germany.
Rietdorf
Jens
J
Pepperkok
Rainer
R
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D005057:Eukaryotic Cells; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence / Q000379:methods*; D038761:Photobleaching; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013057:Spectrum Analysis / Q000295:instrumentation
D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins