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      Working with Commercially Available Quantum Dots for Immunofluorescence on Tissue Sections

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          Abstract

          Quantum dots are semiconductor fluorescent nanocrystals that exhibit excellent characteristics compared with more commonly used organic fluorescent dyes. For many years quantum dot conjugated products have been available in multiple forms for fluorescence imaging of tissue sections under the trademark name Qdot®. They have much increased brightness, narrow emission spectrum, large Stokes shift and photostability compared with conventional organic fluorescent dyes, which together make them the fluorophores of choice for demanding requirements. Vivid Qdots are recent replacements for original Qdots, modified to improve brightness, however this has affected the fluorescence stability in commonly used conditions for immunohistochemistry. We present here our investigation of the stability of original and Vivid Qdots in solution and in immunohistochemistry, highlight the potential pitfalls and propose a protocol for stable and reliable multiplex staining with current commercially available original and Vivid Qdots.

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          Most cited references13

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          Biological applications of quantum dots.

          Quantum dots (QDs) are a novel class of inorganic fluorophore which are gaining widespread recognition as a result of their exceptional photophysical properties. They are rapidly being applied to existing and emerging technologies, and could have an important role in many areas. Significant challenges remain, however, which must be understood and more fully defined before they can be widely validated. This review provides on overview of QD technology, covering QD characteristics, synthesis methods, and the applications in which they have been put to use. The influence of synthesis methods on QD characteristics and their subsequent suitability to different applications is discussed, and a broad outline of the technologies into which they have been incorporated is presented, and the relative merits and weaknesses of their incorporation are evaluated. The potential for further development, and inclusion in other technologies is also discussed, and barriers restricting further progress specified, particularly with regard to the poorly understood surface chemistry of QDs, the potential for alteration of function of biological molecules when complexed with QDs, and on a larger scale the significant potential for cytotoxicity both in vitro and in vivo.
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            Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates.

            Alexa 350, Alexa 430, Alexa 488, Alexa 532, Alexa 546, Alexa 568, and Alexa 594 dyes are a new series of fluorescent dyes with emission/excitation spectra similar to those of AMCA, Lucifer Yellow, fluorescein, rhodamine 6G, tetramethylrhodamine or Cy3, lissamine rhodamine B, and Texas Red, respectively (the numbers in the Alexa names indicate the approximate excitation wavelength maximum in nm). All Alexa dyes and their conjugates are more fluorescent and more photostable than their commonly used spectral analogues listed above. In addition, Alexa dyes are insensitive to pH in the 4-10 range. We evaluated Alexa dyes compared with conventional dyes in applications using various conjugates, including those of goat anti-mouse IgG (GAM), streptavidin, wheat germ agglutinin (WGA), and concanavalin A (ConA). Conjugates of Alexa 546 are at least twofold more fluorescent than Cy3 conjugates. Proteins labeled with the Alexa 568 or Alexa 594 dyes are several-fold brighter than the same proteins labeled with lissamine rhodamine B or Texas Red dyes, respectively. Alexa dye derivatives of phalloidin stain F-actin with high specificity. Hydrazide forms of the Alexa dyes are very bright, formaldehyde-fixable polar tracers. Conjugates of the Alexa 430 (ex 430 nm/em 520 nm) and Alexa 532 (ex 530 nm/em 548 nm) fluorochromes are spectrally unique fluorescent probes, with relatively high quantum yields in their excitation and emission wavelength ranges.
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              Tuning the optical and electronic properties of colloidal nanocrystals by lattice strain.

              Strain can have a large influence on the properties of materials at the nanoscale. The effect of lattice strain on semiconductor devices has been widely studied, but its influence on colloidal semiconductor nanocrystals is still poorly understood. Here we show that the epitaxial deposition of a compressive shell (ZnS, ZnSe, ZnTe, CdS or CdSe) onto a soft nanocrystalline core (CdTe) to form a lattice-mismatched quantum dot can dramatically change the conduction and valence band energies of both the core and the shell. In particular, standard type-I quantum-dot behaviour is replaced by type-II behaviour, which is characterized by spatial separation of electrons and holes, extended excited-state lifetimes and giant spectral shifts. Moreover, the strain induced by the lattice mismatch can be used to tune the light emission--which displays narrow linewidths and high quantum yields--across the visible and near-infrared part of the spectrum (500-1,050 nm). Lattice-mismatched core-shell quantum dots are expected to have applications in solar energy conversion, multicolour biomedical imaging and super-resolution optical microscopy.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                29 September 2016
                2016
                : 11
                : 9
                : e0163856
                Affiliations
                [1 ]University of Edinburgh, Deanery of Molecular Genetics and Public Health Sciences, Department of Pathology, Queen's Medical Research Institute, 47 Little France Crescent, Edinburgh, EH16 4TJ, United Kingdom
                [2 ]University of Edinburgh, Edinburgh Medical School, Centre for Inflammation Research, Queen's Medical Research Institute, 47 Little France Crescent, Edinburgh, EH16 4TJ, United Kingdom
                [3 ]University of Edinburgh, Deanery of Molecular Genetics and Public Health Sciences, Department of Pathology, Royal Infirmary of Edinburgh, 51 Little France Crescent, Edinburgh, EH16 4SA, United Kingdom
                Tohoku University, JAPAN
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                • Conceptualization: SP.

                • Data curation: SP RK.

                • Formal analysis: SP CB.

                • Funding acquisition: SP DK.

                • Investigation: RK SP.

                • Methodology: SP CB.

                • Project administration: SP DK.

                • Resources: CB DK.

                • Supervision: SP CB.

                • Validation: SP CB RK.

                • Visualization: SP.

                • Writing – original draft: SP.

                • Writing – review & editing: CB.

                Author information
                http://orcid.org/0000-0002-0140-7115
                Article
                PONE-D-16-16178
                10.1371/journal.pone.0163856
                5042461
                27685858
                936cb921-c564-442d-acdf-9c4d4226321d
                © 2016 Prost et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 21 April 2016
                : 15 September 2016
                Page count
                Figures: 9, Tables: 3, Pages: 23
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100000589, Chief Scientist Office;
                Award ID: ETM189
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100000848, University Of Edinburgh;
                Award ID: Moray Endowment Fund
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100000848, University Of Edinburgh;
                Award ID: GR01858
                Award Recipient :
                The work was supported by a project grant from the CSO (Chief Scientist Office Scotland, http://www.cso.scot.nhs.uk/) EMT/189 to DK, CB, and SP, a Moray Endowment Fund award to SP (University of Edinburgh, http://www.ed.ac.uk)), and an Innovation grant (GR01858) from the University of Edinburgh Development Trust. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Organisms
                Animals
                Vertebrates
                Amniotes
                Mammals
                Equines
                Asses
                Research and Analysis Methods
                Imaging Techniques
                Fluorescence Imaging
                Research and Analysis Methods
                Specimen Preparation and Treatment
                Staining
                Research and Analysis Methods
                Model Organisms
                Animal Models
                Rabbits
                Biology and Life Sciences
                Organisms
                Animals
                Vertebrates
                Amniotes
                Mammals
                Rabbits
                Research and Analysis Methods
                Specimen Preparation and Treatment
                Staining
                Immunostaining
                Engineering and Technology
                Nanotechnology
                Quantum Dots
                Research and Analysis Methods
                Immunologic Techniques
                Immunoassays
                Immunofluorescence
                Research and Analysis Methods
                Specimen Preparation and Treatment
                Staining
                Immunofluorescence Staining
                Custom metadata
                All data are within the paper and its Supporting Information files.

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                Uncategorized

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