title: Choice of Illumination System & Fluorophore for Multiplex Immunofluorescence on FFPE Tissue Sections.
abstract: The recent availability of novel dyes and alternative light sources to facilitate complex tissue immunofluorescence studies such as multiplex labelling has not been matched by reports critically evaluating the considerations and relative benefits of these new tools, particularly in combination. Product information is often limited to wavelengths used for older fluorophores (FITC, TRITC & corresponding Alexa dyes family). Consequently, novel agents such as Quantum dots are not widely appreciated or used, despite highly favourable properties including extremely bright emission, stability and potentially reduced tissue autofluorescence at the excitation wavelength. Using spectral analysis, we report here a detailed critical appraisal and comparative evaluation of different light sources and fluorophores in multiplex immunofluorescence of clinical biopsy sections. The comparison includes mercury light, metal halide and 3 different LED-based systems, using 7 Qdots (525, 565, 585, 605, 625, 705), Cy3 and Cy5. We discuss the considerations relevant to achieving the best combination of light source and fluorophore for accurate multiplex fluorescence quantitation. We highlight practical limitations and confounders to quantitation with filter-based approaches.
authors.affiliation: Department of Pathology, University of Edinburgh, Deanery of Molecular Genetics and Public Health Sciences, Queen's Medical Research Institute, Edinburgh, Scotland, United Kingdom.
authors.forename: Sandrine
authors.identifier: http://orcid.org/0000-0002-0140-7115
authors.initials: S
authors.lastname: Prost
authors.affiliation: Department of Pathology, University of Edinburgh, Deanery of Molecular Genetics and Public Health Sciences, Queen's Medical Research Institute, Edinburgh, Scotland, United Kingdom.
authors.forename: Ria E B
authors.initials: RE
authors.lastname: Kishen
authors.affiliation: Centre for Inflammation Research, Queen's Medical Research Institute, University of Edinburgh, Edinburgh Medical School, Edinburgh, Scotland, United Kingdom.
authors.forename: David C
authors.initials: DC
authors.lastname: Kluth
authors.affiliation: Department of Pathology, University of Edinburgh, Royal Infirmary of Edinburgh, Edinburgh, Scotland, United Kingdom.
authors.forename: Christopher O C
authors.initials: CO
authors.lastname: Bellamy
mesh_terms: D005456:Fluorescent Dyes / Q000737:chemistry*; D006219:Halogens / Q000737:chemistry; D008670:Metals / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000295:instrumentation*
chemical_list: D005456:Fluorescent Dyes; D006219:Halogens; D008670:Metals