Quantum dots – a versatile tool in plant science?
© Müller et al; licensee BioMed Central Ltd. 2006
Received: 10 February 2006
Accepted: 15 June 2006
Published: 15 June 2006
An optically stable, novel class of fluorophores (quantum dots) for in situ hybridisation analysis was tested to investigate their signal stability and intensity in plant chromosome analyses. Detection of hybridisation sites in situ was based on fluorescence from streptavidin-linked inorganic crystals of cadmium selenide. Comparison of quantum dots (QDs) with conventional detection systems (Alexa 488) in immunolabeling experiments demonstrated greater sensitivity than the conventional system. In contrast, detection of QDs in in situ hybridisation of several plant chromosomes, using several high-copy sequences, was less sensitve than Alexa 488. Thus, semiconductor nanocrystal fluorophores are more suitable for immunostaining but not for in situ hybridisation of plant chromosomes.
Quantum dots (QDs) have been introduced as a promising new tool in life sciences, because of their unique optical properties . They are highly stable during excitation and have characteristic absorption and emission spectra . The emission peak of these nanoparticles is comparatively narrow and the dots fluoresce brighter than organic fluorescent dyes. Thus particle visibility is enhanced and weaker laser intensity is required for the imaging process. QDs can be excited using different wavelengths from UV up to the emission wavelength. Hence it is possible to excite simultaneously QDs emitting at different wavelengths potentially facilitating a simpler handling of multicolor-labelled samples.
Initial attempts to synthesize semiconductor crystals resulted in QDs trapped in glass [2–4]. Later, nanoparticles were developed that could be dispersed in various solvents and whose surface could be derivatized. After hydrophilic coating of QDs with mercaptoacidic acid, dehydrolipoic acid or other reagents, nanocrystals became applicable in biology . The core of the quantum dot particle is composed of a mixture of cadmium and selenide. This sphere, having a diameter of 20 to 55 Å , is coated with 1–2 monolayers of ZnS measuring 3,1 Å.
Proteins, antibodies, DNA or other molecules of interest can be attached to QDs allowing a wide range of applications in life sciences . The complete QD-streptavidin conjugate has a diameter of 10 to 15 nm . Hence quantum dots have been employed in live cell imaging [1, 9], diagnostic and therapeutic purposes , immunohistochemistry  and in fluorescence in situ hybridisation (FISH) experiments [12, 13]. However, until now there have been no reports of applications of QDs in plant research [14, 15]. In order to test whether the application of nanoparticle techniques could improve the sensitivity of in situ hybridisation on plant chromosomes, we conducted a range of comparative test experiments. In addition QDs were employed for immunolabelling of tissue sections.
Materials and methods
For in situ hybridisation of young seedlings of Allium fistulosum samples were pre-treated in iced water for 24 h, fixed in ethanol-glacial acetic acid (3:1, v/v) for 2 days at 4°C and stored at 4°C in 70% ethanol. The ethanol/acetic acid-fixed material was prepared as described in . Alternatively, root meristems were fixed for 30 min in freshly prepared 4% (w/v) formaldehyde solution containing phosphate-buffered saline (PBS, pH 7.3), washed for 45 min in PBS and digested at 37°C for 25 min in a mixture of 2.5% pectinase, 2.5% cellulase Onozuka R-10 and 2.5% pectolyase Y-23 (w/v) dissolved in PBS prior to slide preparation.
Fluorescence in situ hybridisation (FISH)
The generation of probes specific for the A. fistulosum non-coding satellite sequence  was performed as described by . A plasmid VER17  encoding part of the 18S, the 5.8S, most of the 25S and the internal transcribed spacers of Vicia faba 45S rRNA, was used as a rDNA-specific probe.
In situ hybridisation probes were labelled by nick translation with digoxigenin-11-dUTP or biotin-16-dUTP. FISH was carried out according to . For combined probing of rDNA and non-coding satellite DNA, in situ hybridisation was performed using 20 ng of digoxigenin-labeled 45S rDNA and 20 ng of biotin-labelled satellite DNA per slide. Hybridisation sites of the digoxigenin- or biotin-labelled probes were detected using the conventional detection systems, anti-digoxigenin-rhodamine antibody, or streptavidin-Alexa 488 respectively, each at a concentration of 2 μg/ml. In parallel, hybridisation sites of the biotin-labelled probe were detected by using 20 pM QD 565 streptavidin conjugate (Quantum Dot Corporation, USA). The incubation times were 1 h for Alexa 488 and Rhodamine, each, and 2 h for QD565. Working solutions of QDs and antibodies were prepared either in 4 × SSC,1% BSA, 0.1% Tween 20 or Borate buffer (50 mM boric acid H3BO3, pH 6.0 or 7.0). After final washing steps and dehydration, the tissues were mounted in antifade medium containing 10μg/ml DAPI. Fluorescence signals were recorded electronically with a confocal Laser-Scanning-Microscope LSM 510 META (Carl Zeiss Jena GmbH, Jena, Germany) by using laser line 488 for Alexa 488, 543 for rhodamine and 364 for DAPI and QD 565 excitation. Additionally a cooled CCD-camera attached to a standard fluorescence microscope (BX61, Olympus) was used The image manipulations were performed with the program Adobe Photoshop.
Immunolabelling of sectioned material
One mm2 leaf sections of Zea mays were fixed for 3 h at room temperature in 50 mM cacodylate buffer (pH 7.2), containing 0.5% (v/v) glutaraldehyde and 2.0% (v/v) formaldehyde after short vacuum-infiltration. After the fixation the samples were dehydrated in stepwise fashion by adding progressively increasing concentrations of ethanol and concomitantly lowering the temperature (PLT) using an automated freeze substitution unit (AFS, Leica, Benzheim, Germany). The steps used were as follows: 30% (v/v), 40% (v/v) and 50% (v/v) ethanol for 1 h each at 4°C; 60% (v/v) and 75% (v/v) ethanol for 1 h each at -15°C; 90% (v/v) ethanol and two times 100% (v/v) ethanol for 1 h each at -35°C. The samples were subsequently infiltrated with Lowycryl HM20 resin (Plano GmbH, Marburg, Germany) by incubating them in the following mixtures: 33% (v/v), 50% (v/v) and 66% (v/v) HM 20 resin in ethanol for 4 h each and then 100% (v/v) HM 20 overnight. Samples were transferred into gelatine capsules, incubated for 3 h in fresh resin and polymerized at 35°C for 3 days under indirect UV light. 0.5 μm thick sections of the embedded plant tissue were cut with a diamond knife using an Ultramicrotome (Leica Microsystems AG, Wetzlar, Germany) and mounted on slides at 60°C. These sections were washed for 3 × 5 minutes in PBS + 1% BSA at room temperature (RT) and blocked for 20 minutes in PBS + 3% BSA at RT. An antibody from rabbit against CF1 (catalytic portion of the chloroplast H+-ATP synthase), against chloroplasts , and afterwards an anti- rabbit IgG- Biotin conjugate (30 min, both diluted in PBS + 1% BSA, RT) was attached. The secondary antibody was detected with the quantum dot 565-streptavidin conjugate. Each step except blocking was followed by washing as previously described.
Transmission electron microscopy
An aliquot of the QD 565 streptavidin conjugate at a concentration of 0.2 nM was pipetted onto a formvar-coated grid. The grids were pre-treated with poly-L-lysine to increase the binding of the particles. After one minute the grid was drained onto paper and one droplet of 4% uranyl acetate was added. After 15 s the draining procedure was repeated and the grids were air dried. Images were recorded using a Zeiss EM 902 A electron microscope (Carl-Zeiss GmbH, Oberkochen, Germany), equipped with a Megaview III CCD camera (Soft Imaging System, Münster, Germany).
Results and discussion
To improve the performance of quantum dots in in situ hybridisation the following strategies were tested: (1) instead of fixation in an ethanol : acetic acid solution, plant material was fixed in freshly prepared 4% parafomaldehyde for 25 min; (2) to increase the accessibility of chromosomes, different pepsin treatments were used and nuclei were prepared without cytoplasm and (3) 50 mM borate buffer (at pH 6.0 or pH 7.0) was used instead of 2 × SSC. In addition, (4) the concentration of the QD working solution was increased up to ten-fold which resulted in strong background fluorescence (data not shown). (5) Hybridisation of plant chromosomes using the same conditions as those published for mammalian chromosomes using quantum dot-based detection of in situ hybridised probes  were also tried out. Although a number of different possibilities were tested, none of these changes resulted in significantly improved quantum dot-based in situ hybridisation signals in plants. Further, no improvement in in situ hybridisation site detection was obtained with a QD 605 streptavidin conjugate or by using a rabbit anti-biotin antibody detected by a QD 565 anti-Rabbit IgG conjugate (both: Quantum Dot Corporation, USA). Additionally, similar results were obtained for detection of labeled 45S rDNA on chromosomes of Arabidopsis thaliana and Nicotiana tabacum using quantum dots.
In summary, while quantum dot-based immunodetection is a promising new tool in plant science, it seems that problems of handling the nanocrystals occur in FISH experiments with plant chromosomes. We suggest that these large semiconductor nanocrystal fluorophores suffer from steric hinderances which preclude their use in in situ hybridisation to plant chromatin.
I would like to thank Twan Rutten for general help and Jeremy Timmis for critical reading of the manuscript. We are grateful to Bernhard Claus, Katrin Kumke and Sylvia Marschner for excellent technical assistance.
Certain commercial entities, equipment, or materials may be identified in this paper in order to describe an experimental procedure or concept adequately. Such identification is not intended to imply recommendation or endorsement by the National Institute of Standards and Technology, nor is it intended to imply that the entities, materials, or equipment are necessarily the best available for the purpose.
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