Chromosome Research

, Volume 17, Issue 4, pp 519–530

Quantum dots as new-generation fluorochromes for FISH: an appraisal

  • Dimitris Ioannou
  • Helen G. Tempest
  • Benjamin M. Skinner
  • Alan R. Thornhill
  • Michael Ellis
  • Darren K. Griffin
Article

Abstract

In the field of nanotechnology, quantum dots (QDs) are a novel class of inorganic fluorochromes composed of nanometre-scale crystals made of a semiconductor material. Given the remarkable optical properties that they possess, they have been proposed as an ideal material for use in fluorescent in-situ hybridization (FISH). That is, they are resistant to photobleaching and they excite at a wide range of wavelengths but emit light in a very narrow band that can be controlled by particle size and thus have the potential for multiplexing experiments. The principal aim of this study was to compare the potential of QDs against traditional organic fluorochromes in both indirect (i.e. QD-conjugated streptavidin) and direct (i.e. synthesis of QD-labelled FISH probes) detection methods. In general, the indirect experiments met with a degree of success, with FISH applications demonstrated for chromosome painting, BAC mapping and use of oligonucleotide probes on human and avian chromosomes/nuclei. Many of the reported properties of QDs (e.g. brightness, ‘blinking’ and resistance to photobleaching) were observed. On the other hand, signals were more frequently observed where the chromatin was less condensed (e.g. around the periphery of the chromosome or in the interphase nucleus) and significant bleed-through to other filters was apparent (despite the reported narrow emission spectra). Most importantly, experimental success was intermittent (sometimes even in identical, parallel experiments) making attempts to improve reliability difficult. Experimentation with direct labelling showed evidence of the generation of QD-DNA constructs but no successful FISH experiments. We conclude that QDs are not, in their current form, suitable materials for FISH because of the lack of reproducibility of the experiments; we speculate why this might be the case and look forward to the possibility of nanotechnology forming the basis of future molecular cytogenetic applications.

Keywords

quantum dot nanotechnology FISH chromosome painting semiconductor 

Abbreviations

BAC(s)

bacterial artificial chromosome(s)

BSA

bovine serum albumin

DAPI

4′,6-diamidino-2-phenylindole

ddH2O

double-distilled water

DS

dextran sulfate

DOP

degenerate oligo primed

DTT

dithiothreitol

dUTP

2′-deoxyuridine 5′-triphosphate

FA

formamide

FISH

fluorescence in-situ hybridization

FITC

fluorescein isothiocyanate

HFEA

human fertilization and embryology authority

MAA

mercaptoacetic acid

NIR

near infrared

PBS

phosphate-buffered saline

QD

quantum dot

QD-FISH

quantum dot fluorescence in-situ hybridization

RT

room temperature

PCR

polymerase chain reaction

SERT

serotonin transporter protein

SSC

saline sodium citrate

UV

ultraviolet

Supplementary material

10577_2009_9051_Fig8_ESM.jpg (38 kb)
Supplementary Fig. S1

QD585 dissolved in hybridization mix and viewed directly under the microscope using four barrier filters: 525 nm (blue), 565 nm (green), 585 nm (red) and 605 nm (far red but pseudo-coloured purple for the purposes of this figure). The image is a merge of all four filters. The QDs are predominantly red (as would be expected), but a smaller number of green, blue and purple QDs are seen. The discrete appearance of QDs of one or other of the colours indicates there is a mixed population of QDs in each preparation (37.8 KB).

10577_2009_9051_MOESM1_ESM.mpg (118 kb)
Supplementary Movie S2aMovie of QD585 dissolved in hybridization mix and viewed directly under the microscope using 585 nm barrier filter. The phenomenon of ‘blinking’ is clearly seen (MPG 118 KB).
10577_2009_9051_MOESM2_ESM.mpg (128 kb)
Supplementary Movie S2bMovie of QD605 dissolved in hybridization mix and viewed directly under the microscope using 605nm barrier filter. The phenomenon of ‘blinking’ is clearly seen (MPG 128 KB).

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Dimitris Ioannou
    • 1
  • Helen G. Tempest
    • 1
    • 2
  • Benjamin M. Skinner
    • 1
  • Alan R. Thornhill
    • 1
    • 2
  • Michael Ellis
    • 3
  • Darren K. Griffin
    • 1
  1. 1.Department of BiosciencesUniversity of KentCanterburyUK
  2. 2.The London Bridge Fertility, Gynaecology and Genetics Centre and Bridge GenomaLondonUK
  3. 3.Digital Scientific UK LtdCambridgeUK

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