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.
Subject(s)
Bacterial Proteins/chemistry , Green Fluorescent Proteins/metabolism , Luminescent Proteins/chemistry , Secretory Vesicles/chemistry , Vesicle-Associated Membrane Protein 2/chemistry , Animals , Astrocytes/metabolism , Biophysics/methods , Hydrogen-Ion Concentration , Mice , Microscopy, Fluorescence/methods , Spectrum Analysis/methodsABSTRACT
The figure of merit for the electron optical performance of carbon-nanotube (CNT) electron sources is presented. This figure is given by the relation between the reduced brightness and the energy spread in the region of stable emission. It is shown experimentally that a CNT electron source exhibits a highly stable emission process that follows the Fowler-Nordheim theory for field emission, fixing the relationship among the energy spread, the current, and the radius. The performance of the CNT emitter under realistic operating conditions is compared with state-of-the-art electron point sources. It is demonstrated that the reduced brightness is a function of the tunneling parameter, a measure of the energy spread at low temperatures, only, independent of the geometry of the emitter.
