Authors

Sandrine Prost, Ria E. B. Kishen, David C. Kluth, ChristopherO. C. Bellamy

Affiliations

1 Department of Pathology, University of Edinburgh, Deanery of Molecular Genetics and Public Health
Sciences, Queen's Medical Research Institute, Edinburgh, Scotland, United Kingdom, 2 Centre for
Inflammation Research, Queen's Medical Research Institute, University of Edinburgh, Edinburgh Medical
School, Edinburgh, Scotland, United Kingdom, 3 Department of Pathology, University of Edinburgh, Royal
Infirmary of Edinburgh, Edinburgh, Scotland, United Kingdom

Application Area

Cell Biology

Immunofluorescence

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.

Extract

“The pE-4000 from CoolLED, with capacity for 16 wavelengths
that can be turned on individually or in groups of 4; secondly, the pE-2 with 2 or 4 LAM.”

Product Associated Features

pE-4000: The universal LED illumination system for research fluorescence, with the broadest spectrum of illumination available from 16 selectable wavelengths, and extensive functionality that sets a new standard for industry.

Diascopic Technique

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Live Cell Issues

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Product Type

pE-4000

Journal

PloS one

Year of Publication

2016

Country of Publication

Italy