Quantitative Colocalisation Imaging: Concepts, Measurements, and Pitfalls

DOI: 10.1007/978-3-540-71331-9_5


Many questions in cell biology and biophysics involve the quantitation of the colocalisation of proteins tagged with different
fluorophores and their interaction. 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 multiband images ambiguous. Traditionally often used in a qualitative manner by simply overlaying
fluorescence images (‘red plus green equals yellow’), multicolour fluorescence is increasingly moving away from static dual-colour
images towards more quantitative studies involving the investigation of dynamical three-dimensional interaction of proteins
tagged with different fluorophores in live cells. Quantifying fluorescence resonance energy transfer efficiency, fluorescence
complementation and colour merging following photoactivation or photoswitching provide related examples in which quantitative
image analysis of multicolour fluorescence is required. Despite its widespread use, reliable standards for evaluating the
degree of spectral overlap in multicolour fluorescence and calculating quantitative colocalisation estimates are missing.
In this chapter, using a number of intuitive yet practical examples, we discuss the factors that affect image quality and
study their impact on the measured degree of colocalisation. We equally compare different pixel-based and object-based descriptors
for analysing colocalisation of spectrally separate fluorescence. Finally, we discuss the use of spectral imaging and linear
unmixing to study the presence in a ‘mixed pixel’ of spectrally overlapping fluorophores and discuss how this technique can
be used to provide quantitative colocalisation information in more complex experimental scenarios in which classic dual- or
triple-colour fluorescence would produce erroneous results.

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    • "All too often, the interpretation becomes subjective and the researcher must consider the likelihood that the low frequency of colocalization is caused by a biological mechanism rather than chance. To answer this question wither greater accuracy, new approaches are being developed for quantitative colocalization, including image crosscorrelation methods and the Manders coefficients (Oheim, 2007; Comeau et al., 2006). Effectively, these methods quantify how often and in how many pixels two different probes are present. "
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    ABSTRACT: Advances in microscopy and fluorescent probes provide new insight into the nanometer-scale biochemistry governing the interactions between eukaryotic cells and pathogens. When combined with mathematical modelling, these new technologies hold the promise of qualitative, quantitative and predictive descriptions of these pathways. Using the light microscope to study the spatial and temporal relationships between pathogens, host cells and their respective biochemical machinery requires an appreciation for how fluorescent probes and imaging devices function. This review summarizes how live cell fluorescence microscopy with common instruments can provide quantitative insight into the cellular and molecular functions of hosts and pathogens.
    Cellular Microbiology 02/2009; 11(4):540-50. DOI:10.1111/j.1462-5822.2009.01283.x · 4.92 Impact Factor
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    • "Its absorption and emission spectra are both red-shifted compared to that of EGFP with peaks at 488 nm and 507–509 nm, respectively [11], Fig. 1A. Using the spectral separability index X ijk [6], we estimated that for an equimolar mixture of Calcium Ruby and EGFP, below 1% of the fluorescence detected in the EGFP and Calcium Ruby channel are due to cross-talk, see Supplementary Table T1. Indeed, no crossexcitation or fluorescence bleed-through were detectable when mouse embryonic spinal cord motoneurons expressing EGFP controlled by regulatory elements of the glycine transporter 2 (GlyT2) gene were whole-cell patch-clamped and loaded with 30 M Calcium Ruby in the pipette and imaged in dual-color epifluorescence, Fig. 1B. "
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    ABSTRACT: Dual-color imaging of acridine orange (AO) and EGFP fused to a vesicular glutamate transporter or the vesicle-associated membrane proteins 2 or 3 has been used to visualize a supposedly well-defined subpopulation of glutamatergic astrocytic secretory vesicles undergoing regulated exocytosis. However, AO metachromasy results in the concomitant emission of green and red fluorescence from AO-stained tissue. Therefore, the question arises whether AO and EGFP fluorescence can be distinguished reliably. We used evanescent-field imaging with spectral fluorescence detection as well as fluorescence lifetime imaging microscopy to demonstrate that green fluorescent AO monomers inevitably coexist with red fluorescing AO dimers, at the level of single astroglial vesicles. The green monomer emission spectrally overlaps with that of EGFP and produces a false apparent colocalization on dual-color images. On fluorophore abundance maps calculated from spectrally resolved and unmixed single-vesicle spectral image stacks, EGFP is obscured by the strong green monomer fluorescence, precluding the detection of EGFP. Hence, extreme caution is required when deriving quantitative colocalization information from images of dim fluorescing EGFP-tagged organelles colabeled with bright and broadly emitting dyes like AO. We finally introduce FM4-64/EGFP dual-color imaging as a remedy for imaging a distinct population of astroglial fusion-competent secretory vesicles.
    Biophysical Journal 09/2007; 93(3):969-80. DOI:10.1529/biophysj.106.102673 · 3.97 Impact Factor
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