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| Content Provider | Springer Nature Link |
|---|---|
| Author | Nadrigny, Fabien Rivals, Isabelle Hirrlinger, Petra G. Koulakoff, Annette Personnaz, Léon Vernet, Marine Allioux, Myriam Chaumeil, Myriam Ropert, Nicole Giaume, Christian Kirchhoff, Frank Oheim, Martin |
| Copyright Year | 2006 |
| Abstract | Many questions in cell biology and biophysics involve the quantitation of co-localisation and the interaction of proteins tagged with different fluorophores. However, the incomplete separation of the different colour channels due to the presence of autofluorescence, along with cross-excitation and emission “bleed-through” of one colour channel into the other, all combine to render the interpretation of multi-band images ambiguous. Here we introduce a new live-cell epifluorescence spectral imaging and linear unmixing technique for classifying resolution-limited point objects containing multiple fluorophores. We demonstrate the performance of our technique by detecting, at the single-vesicle level, the co-expression of the vesicle-associated membrane protein, VAMP-2 (also called synaptobrevin-2), linked to either enhanced green fluorescent protein (EGFP) or citrine [a less pH-sensitive variant of enhanced yellow fluorescent protein (EYFP)], in mouse cortical astrocytes. In contrast, the co-expression of VAMP-2-citrine and the lysosomal transporter sialine fused to EGFP resulted in little overlap. Spectral imaging and linear unmixing permit us to fingerprint the expression of spectrally overlapping fluorescent proteins on single secretory organelles in the presence of a spectrally broad autofluorescence. Our technique provides a robust alternative to error-prone dual- or triple colour co-localisation studies. |
| Starting Page | 533 |
| Ending Page | 547 |
| Page Count | 15 |
| File Format | |
| ISSN | 01757571 |
| Journal | European Biophysics Journal |
| Volume Number | 35 |
| Issue Number | 6 |
| e-ISSN | 14321017 |
| Language | English |
| Publisher | Springer-Verlag |
| Publisher Date | 2006-03-28 |
| Publisher Place | Berlin, Heidelberg |
| Access Restriction | One Nation One Subscription (ONOS) |
| Subject Keyword | Spectral imaging Linear unmixing Fluorescence microscopy Total internal reflection Exocytosis Protein expression Co-localisation Biophysics/Biomedical Physics Neurobiology Animal Physiology Plant Physiology Cell Biology Biochemistry |
| Content Type | Text |
| Resource Type | Article |
| Subject | Medicine Biophysics |
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