Nanotechnology and molecular cytogenetics: the future has not yet arrived

Abstract

Quantum dots (QDs) are a novel class of inorganic fluorochromes composed of nanometer-scale crystals made of a semiconductor material. They are resistant to photo-bleaching, have narrow excitation and emission wavelengths that can be controlled by particle size and thus have the potential for multiplexing experiments. Given the remarkable optical properties that quantum dots possess, they have been proposed as an ideal material for use in molecular cytogenetics, specifically the technique of fluorescent in situ hybridisation (FISH). In this review, we provide an account of the current QD-FISH literature, and speculate as to why QDs are not yet optimised for FISH in their current form.

Keywords: FISH; imaging; nanotechnology; quantum dot.

fig. 1

Schematic representation of a QD conjugate.

fig. 2

The size-dependent luminescence of quantum dots. Larger QDs have narrow band gaps (red QD, b) comparing to small QDs (blue QD, b). In the example discussed, the 5.5 QD emits orange light (longer wavelength 590 nm), whereas the 2.3 QD emits turquoise light (shorter wavelength 500 nm). Adapted from Jonathan (17).

fig. 3

Comparison of absorption and excitation spectra between FITC (Fluorescein isothiocyanate) (blue) and a CdSe QD (green). Adapted from Bailey et al. (22).

fig. 4

QD520 (supplied by Evident) spotted on to a glass slide, excited by a UV filter and then detected with barrier filters at 525, 565, 585 and 605 nm, respectively. Although under the green barrier filter (525 nm) the brightest fluorescence is observed, significant bleed-through is seen on the other filters indicating that the emission spectrum is not as narrow as is usually purported for QDs.

fig. 5

Successful FISH experiments on human chromosome 1 using biotinylated chromosome 1 paint with Cy3-streptavidin conjugate control (upper) and QD585-streptavidin conjugate (lower). QD585 signals were brighter, though more ‘patchy’ and with a greater amount of background. Adapted from Ioannou et al. (90).

fig. 6

Successful chromosome painting experiment (chromosome 2, tetraploid cell) in chicken, but with signals predominantly around the periphery of the chromosome, giving an impression of a fluorescent ‘sheath’. Adapted from Ioannou et al. (90).

fig. 7

(A) Chromosome painting attempt in human lymphocytes using QD520. No specific signal was seen and the area surrounding the chromosomes had a very high background (left), moreover the background signal bled through into the red channel (right). (B) Attempts to visualise the centromeres of human chromosome 12. There is some evidence of hybridisation and detection but the preparation has a very high background. (C) A bright red signal is seen on every part of the slide apart from the chromosomes! This was another attempt at human chromosome painting for chromosomes 1 and 2.

fig. 8

QD605 dissolved in hybridisation mix and viewed directly under the microscope using four barrier filters: 525 nm (blue), 565 nm, 585 nm (red) and 605 nm (far red but pseudo-coloured purple for the purposes of this figure). The image represents a merge of all four filters. The QDs are predominantly purple (as would be expected), but a smaller number of green, blue and red QDs are seen. The discrete appearance of QDs of one or other of the colours indicates there is a mixed population of QDs. Adapted from Ioannou et al. (90).