QDs versus Alexa: reality of promising tools for immunocytochemistry

Abstract

Background: The unique photonic properties of the recently developed fluorescent semiconductor nanocrystals (QDs) have made them a potential tool in biological research. However, QDs are not yet a part of routine laboratory techniques. Double and triple immunocytochemistries were performed in HeLa cell cultures with commercial CdSe QDs conjugated to antibodies. The optical characteristics, due to which QDs can be used as immunolabels, were evaluated in terms of emission spectra, photostability and specificity.

Results: QDs were used as secondary and tertiary antibodies to detect beta-tubulin (microtubule network), GM130 (Golgi complex) and EEA1 (endosomal system). The data obtained were compared to homologous Alexa Fluor 594 organic dyes. It was found that QDs are excellent fluorochromes with higher intensity, narrower bandwidth values and higher photostability than Alexa dyes in an immunocytochemical process. In terms of specificity, QDs showed high specificity against GM130 and EEA1 primary antibodies, but poor specificity against beta-tubulin. Alexa dyes showed good specificity for all the targets tested.

Conclusion: This study demonstrates the great potential of QDs, as they are shown to have superior properties to Alexa dyes. Although their specificity still needs to be improved in some cases, QDs conjugated to antibodies can be used instead of organic molecules in routine immunocytochemistry.

Figure 1

HRTEM QD characterization. The large image shows a general view of QD 655 dispersion. The small image shows a detail of a single QD 655 cone-like nanocrystal. Its crystalline structure core can be seen. Scale bar = 10 nm.

Figure 2

Fluorescence emission spectra. Spectral profile representing fluorescence intensity versus emission wavelength (500–780 nm) for QD 655-IgG, QD 655-Streptavidin and their Alexa homologues. Excitation wavelength = 488 nm.

Figure 3

Photostability profile. Fluorescence intensity changes of QDs and Alexas during the irradiation period with the 561 nm laser line at maximum power.

Figure 4

CLSM photostability images. Left-hand images correspond to the emission signal of QDs and Alexas conjugated to streptavidin before the irradiation period (t = 0 min) with the 561 nm laser line at its maximum power. Right-hand images show the emission signal of QDs and Alexas at the end of the irradiation period (t = 8 min). Note the loss of fluorescence intensity in the delimited area. Scale bar = 10 μm.

Figure 5

CLSM specificity analysis of β-tubulin labeling. Maximum intensity projections of the distribution of the tubulin network labeled with QDs (top images) show lower specificity than their organic Alexa homologue labeling (bottom images). Scale bar = 10 μm.

Figure 6

CLSM specificity analysis of GM130 and EEA1 labeling. Isosurface representation of the cell shows the nucleus (blue) labeled with Hoechst 33342, Golgi complex (GM130) and endosomal system (EEA1) (red) within a three-dimensional volumetric x-y-z data field. Scale bar = 10 μm.