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

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.

Lysosomes Are the Major Vesicular Compartment Undergoing Ca2+-Regulated Exocytosis from Cortical Astrocytes

“…In some experiments, we used the lower-affinity Ca Quantitative multicolor imaging and colocalization analysis. We used a custom inverted microscope equipped for brightfield as well as polychromatic epifluorescence and through-the-objective (60ϫ; numerical aperture, 1.45) evanescent-field fluorescence excitation (Nadrigny et al, 2006(Nadrigny et al, , 2007. A Polychrome II light source (TILL Photonics) provided monochromatic (18 nm full-width at half-maximum) epifluorescence illumination.…”

“…Controls of the correct targeting of the transfected plasmids were performed with cytoplasmically targeted EGFP (pEGFP-N1; Clontech) (Nadrigny et al, 2006). Neither the number nor the fusion competence of FM-labeled organelles was altered by cell transfection (supplemental Fig.…”

“…Expression was checked the day after transfection by systematically comparing the fluorescence excitation and the five-point emission spectrum of transfected astrocytes with their nonlabeled counterparts from the same preparation (data not shown) (Nadrigny et al, 2006). Controls of the correct targeting of the transfected plasmids were performed with cytoplasmically targeted EGFP (pEGFP-N1; Clontech) (Nadrigny et al, 2006).…”

“…We used as organelle markers the following fusion proteins: vesicular-associated membrane proteins VAMP2 (a gift from J. P. Mothet, Institu Fédératif de Neurobiologie Alfred Fessard, Centre National de la Recherche Scientifique, Université Paris Descartes 9040, Bordeaux, France), VAMP3 (from T. Galli, Institut Jacques Monod, Unité Mixte de Recherche 7592, Paris, France) fused on their C terminus to pEGFP-N1 (Clontech), EGFP-sialin (from B. Gasnier, Institut de Biologie Physico-Chimique, Centre National de la Recherche Scientifique, Unité Propre de Recherche 1929, Paris, France), CD63-EGFP, Ti-VAMP/VAMP7-EGFP, endobrevin/VAMP8-EGFP (all from T. Galli). The exposure to low pH of EGFP in the vesicle lumen affects its intensity but not the spectrum (Nadrigny et al, 2006). VGlut1-Venus and VGlut3-Venus (from E. Herzog, Max Planck Institute for Experimental Medicine, Göttingen, Germany) were transfected as markers of glutamatergic vesicles.…”

“…The total illuminated near-membrane section (1/e 2 -intensity decay) sampled by the evanescent field was on the order of 200 nm. For emission spectral imaging (Nadrigny et al, 2006), five emission spectral images were acquired sequentially on 458 nm excitation (supplemental Fig. 5, available at www.jneurosci.org as supplemental material).…”