Detecting fluorescent protein expression and co-localisation on single secretory vesicles with linear spectral unmixing.
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.
Molecular and Cellular Biophysics of Synaptic Transmission, Laboratory of Neurophysiology and New Microscopies, INSERM U603, CNRS FRE 2500, Université René Descartes (Paris 5), 45 rue des Saints Pères, 75 006, Paris, France. fabien.ropert@univ-paris5.fr
Fabien
F
Nadrigny
Isabelle
I
Rivals
Petra G
PG
Hirrlinger
Annette
A
Koulakoff
Léon
L
Personnaz
Marine
M
Vernet
Myriam
M
Allioux
Myriam
M
Chaumeil
Nicole
N
Ropert
Christian
C
Giaume
Frank
F
Kirchhoff
Martin
M
Oheim
D000818:Animals; D001253:Astrocytes / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry*; D001703:Biophysics / Q000379:methods; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008164:Luminescent Proteins / Q000737:chemistry*; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods; D022142:Secretory Vesicles / Q000737:chemistry*; D013057:Spectrum Analysis / Q000379:methods; D050988:Vesicle-Associated Membrane Protein 2 / Q000737:chemistry*
D001426:Bacterial Proteins; D008164:Luminescent Proteins; D050988:Vesicle-Associated Membrane Protein 2; C485184:enhanced green fluorescent protein; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins