European Biophysics Journal

, Volume 35, Issue 6, pp 533–547 | Cite as

Detecting fluorescent protein expression and co-localisation on single secretory vesicles with linear spectral unmixing

  • Fabien Nadrigny
  • Isabelle Rivals
  • Petra G. Hirrlinger
  • Annette Koulakoff
  • Léon Personnaz
  • Marine Vernet
  • Myriam Allioux
  • Myriam Chaumeil
  • Nicole Ropert
  • Christian Giaume
  • Frank Kirchhoff
  • Martin OheimEmail author
Biophysics Letter


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.


Spectral imaging Linear unmixing Fluorescence microscopy Total internal reflection Exocytosis Protein expression Co-localisation 





Carbonyl cyanide m-chlorophenylhydrazone


Enhanced yellow fluorescent protein


Fluorescence resonance energy transfer


Ethylenediaminetetraacetic acid


Dulbecco’s modified Eagle’s medium


Evanescent field


Enhanced green fluorescent protein


Fetal calf serum


Fluorescent protein


Full-width half maximum


Glial fibrillary acidic protein


Postnatal days 0–1


Phosphate-buffered saline


Photoswitchable cyan FP


Standard deviation


Confidence interval


Spectral imaging and linear unmixing


Total internal reflection fluorescence


Vesicle-associated membrane protein



The authors thank S.L. Shorte for comments on the manuscript and M. Coppey-Moissan for the loan of equipment. Supported by the Institut National de la Santé et de la Recherche Médicale (INSERM) and a joint grant from the Centre National de la Recherche Scientifique (CNRS) and the Ministère National de la Recherche et de la Technologie (AC DRAB no. 03/93-2003) to CG and MO and a joint Max-Planck/INSERM AMIGO grant “Molecular Bases of Astroglial Signalling under Physiological and Pathological Conditions” (to CG, MO and FK). FN was the recipient of a Ph.D. Ministère National de la Recherche et de la Technologie studentship, PGH was financed by a Max-Planck postgraduate Ph.D. studentship.

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Copyright information

© EBSA 2006

Authors and Affiliations

  • Fabien Nadrigny
    • 1
  • Isabelle Rivals
    • 2
  • Petra G. Hirrlinger
    • 3
  • Annette Koulakoff
    • 4
  • Léon Personnaz
    • 2
  • Marine Vernet
    • 1
  • Myriam Allioux
    • 1
  • Myriam Chaumeil
    • 1
  • Nicole Ropert
    • 1
  • Christian Giaume
    • 4
  • Frank Kirchhoff
    • 3
  • Martin Oheim
    • 1
    Email author
  1. 1.Molecular and Cellular Biophysics of Synaptic Transmission, Laboratory of Neurophysiology and New Microscopies, INSERM U603, CNRS FRE 2500Université René Descartes (Paris 5)ParisFrance
  2. 2.Applied Statistics GroupEcole Supérieure de Physique et Chimie Industrielles (ESPCI)ParisFrance
  3. 3.Department of NeurogeneticsMax-Planck Institute for Experimental MedicineGöttingenGermany
  4. 4.Laboratoire deNeuropharmacologie INSERM U587Collège de FranceParisFrance

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