Quantitative Colocalisation Imaging: Concepts, Measurements, and Pitfalls

DOI: 10.1007/978-3-540-71331-9_5

ABSTRACT 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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    ABSTRACT: Quantum Dots (QDs) are semiconductor nanocrystals (1~20 nm) exhibiting distinctive photoluminescence (PL) properties due to the quantum confinement effect. Having many advantages over organic dyes, such as broad excitation and resistance to photobleaching, QDs are widely used in bioapplications as one of most exciting nanobiotechnologies. To date, most commercial QDs are synthesized through the traditional organometallic method and contain toxic elements, such as cadmium, lead, mercury, arsenic, etc. The overall goal of this thesis study is to develop an aqueous synthesis method to produce nontoxic quantum dots with strong emission and good stability, suitable for biomedical imaging applications. Firstly, an aqueous, simple, environmentally friendly synthesis method was developed. With cadmium sulfide (CdS) QDs as an example system, various processing parameters and capping molecules were examined to improve the synthesis and optimize the PL properties. The obtained water soluble QDs exhibited ultra small size (~5 nm), strong PL and good stability. Thereafter, using the aqueous method, the zinc sulfide (ZnS) QDs were synthesized with different capping molecules, i.e., 3-mercaptopropionic acid (MPA) and 3-(mercaptopropyl)trimethoxysilane (MPS). Especially, via a newly developed capping molecule replacement method, the present ZnS QDs exhibited bright blue emission with a quantum yield of 75% and more than 60 days lifetime in the ambient conditions. Two cytotoxicity tests with human endothelial cells verified the nontoxicity of the ZnS QDs by cell counting with Trypan blue staining and fluorescence assay with Alamar Blue. Taking advantage of the versatile surface chemistry, several strategies were explored to conjugate the water soluble QDs with biomolecules, i.e., antibody and streptavidin. Accordingly, the imaging of Salmonella t. cells and biotinylated microbeads has been successfully demonstrated. In addition, polyethylenimine (PEI)-QDs complex was formed and delivered into PC12 neuronal cells for intracellular imaging with uniform distribution. The water soluble QDs were also embedded in electrospun polymer fibers as fluorescent nanocomposite. In summary, the ease of aqueous processing and the excellent PL properties of the nontoxic water soluble ZnS QDs provide great potential for various in vivo applications.

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May 30, 2014