ArticleEuropean biophysics journal : EBJ2006
Detecting fluorescent protein expression and co-localisation on single secretory vesicles with linear spectral unmixing.
Article in European biophysics journal : EBJ, 2006. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 17 papers.
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Who cites it
17 citing papers in PubMed, 39 citations in OpenAlex.
- High-speed wide-field fluorescence lifetime imaging for intraoperative tumor visualization and in vivo multiplexing.Biomedical optics express · 2025Article
- Comparison of fluorescence lifetime and multispectral imaging for quantitative multiplexing in biological tissue.Biomedical optics express · 2022Article
- Normalization of optical fluence distribution for three-dimensional functional optoacoustic tomography of the breast.Journal of biomedical optics · 2022Article
- Robust blind spectral unmixing for fluorescence microscopy using unsupervised learning.PloS one · 2019Article
- Induction of hippocampal long-term potentiation increases the morphological dynamics of microglial processes and prolongs their contacts with dendritic spines.Scientific reports · 2016Article
- Astrocyte VAMP3 vesicles undergo Ca2+ -independent cycling and modulate glutamate transporter trafficking.The Journal of physiology · 2015Article
- Quantitative measurement of cancer tissue biomarkers in the lab and in the clinic.Laboratory investigation; a journal of technical methods and pathology · 2015Review
- New tools for investigating astrocyte-to-neuron communication.Frontiers in cellular neuroscience · 2013Review
- Lack of evidence for vesicular glutamate transporter expression in mouse astrocytes.The Journal of neuroscience : the official journal of the Society for Neuroscience · 2013Article
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- Optogenetic activation of LiGluR-expressing astrocytes evokes anion channel-mediated glutamate release.The Journal of physiology · 2012Article
- Live cell fluorescence microscopy to study microbial pathogenesis.Cellular microbiology · 2009Review
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- Lysosomes are the major vesicular compartment undergoing Ca2+-regulated exocytosis from cortical astrocytes.The Journal of neuroscience : the official journal of the Society for Neuroscience · 2008Article
- Three-dimensional FRET reconstruction microscopy for analysis of dynamic molecular interactions in live cells.Biophysical journal · 2008Article
- The potential of high-content high-throughput microscopy in drug discovery.British journal of pharmacology · 2007Review
- Systematic colocalization errors between acridine orange and EGFP in astrocyte vesicular organelles.Biophysical journal · 2007Article
Corrections and comments
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Authors and funding
12 authors at 6 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
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.
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