To test whether the unique optical properties of QDs could be used to improve the signal intensity and stability of in situ hybridised probes, chromosomes were hybridised with different types of high copy sequences. Using the conventional detection system (Alexa 488, rhodamine), strong hybridisation signals of the biotin-labelled A. fistulosum satellite or digoxigenin-labelled 45S rDNA were detectable at the expected chromosomal sites (Fig. ). After confirming the suitability of the probes and of the hybridisation procedure, the same conditions were used to test the suitability of quantum dot technology for detection of in situ hybridised probes. Therefore, instead of detecting the biotinylated probe by streptavidin-Alexa 488 we employed a QD 565-streptavidin conjugate. However, when visualised by both types of fluorescence microscopes, QDs revealed only very weak hybridisation signals, while the anti-digoxigenin-rhodamine detected 45S rDNA control signals were always clearly visible (Fig. ).
Figure 1 (a) Somatic chromosome and nuclei of Allium fistulosum after DAPI staining and fluorescence in situ hybridization with labelled non-coding satellite sequences (green signals for Alexa 488) and 45S rDNA (red signals for rhodamine) using conventional detection (more ...)
To monitor the quality of the QD conjugate employed for the in situ
hybridisation experiments, the same QDs were used for immunolabelling of leaf sections of Zea mays
with the anti-CF1 antibody. Confocal laser scanning microscopy imaging revealed a strong and CF1-specific immunolabelling of chloroplasts (Fig. ). The emission spectrum peaks at 565 nm, hence demonstrating the functionality of the QDs tested for immunohistochemistry (Fig ). In parallel, anti-CF1 signals were detected using an Alexa 488 streptavidin conjugate as control. To compare the stability of Alexa 488 and QD 565-signals, both probes were laser scanned repeatedly 100 times. Immunofluorescence of nanocrystal fluorophores was significantly brighter and more photostable (Fig. ) than the organic fluorophore Alexa 488, as previously demonstrated in similar applications [2
Figure 2 (a) Immunolabelled section of a resin-embedded Zea mays leaf using a CF1- antibody and subsequent signal detection by a combination of an anti-rabbit IgG- Biotin/quantum dot 565-streptavidin conjugate. Note the strongly labelled chloroplasts. White cross (more ...)
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 [13
] 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.
Why was the signal detection of in situ
hybridised probes via quantum dots comparatively lower on the chromosomes of plants when the application of this technique to mammalian chromosomes was efficient? We suspect that lack of labels on chromosomes could be due to sterical hinderance of the rather large quantum dots into the more densely packed plant chromatin, compared to animal chromatin [22
]. Further, the formamide treatment required for in situ
hybridisation of chromosomes causes considerable changes in the chromatin structure [23
], which could negatively influence the accessibility of chromatin. Measurement of the size of the quantum dots revealed a diameter of 15 nm per dot (Fig. ), whereas that of Alexa 488-streptavidin is only 0.6 nm, suggesting a much greater capability to penetrate chromatin. Notably, a size dependence on the accessibility of immunoreactants in fixed chromatin was discussed for immunogold markers [24
]. These results suggested that, for sterical reasons, the immunolabelling of plant chromosomes could be performed with 1.4 nm Nanogold-labelled antibodies, but not with 10 nm gold-labelled antibodies.
Transmission electron micrograph of the QD 565 streptavidin conjugate after negative staining reveals a particle size of 15 nm. Further enlarged quantum dots are shown in square inset.
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.