Quantum Dots for Live-Cell Imaging

Qdot® Conjugates Go the Distance to Monitor Lipid Raft Microdomains

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Time-lapse imaging of live cells labeled with fluorescent dyes is a powerful technique for interrogating cell biology. As compared with organic fluorophores, the bright and photostable Qdot® conjugates can provide higher-content imaging by enabling more frequent image acquisitions with shorter exposure times. Additionally, our recently improved Qdot® conjugates now offer reduced intensity differences between the different colors, simplifying multiplex applications when using violet-light excitation.

Lipid Raft Labeling With Qdot® Conjugates

Lipid rafts are detergent-insoluble, sphingolipid- and cholesterol-rich membrane microdomains that form lateral assemblies in the plasma membrane. They have been shown to play a role in a variety of cellular processes, including the compartmentalization of cell signaling events, the regulation of apoptosis, and the intracellular trafficking of certain membrane proteins and lipids, as well as in the infectious cycles of several viruses and bacterial pathogens [1–3].To demonstrate the advantages of Qdot® conjugates for time-lapse imaging, we labeled lipid rafts using either a direct Alexa Fluor® dye conjugate of cholera toxin subunit B (CT-B) or a biotinylated CT-B in conjunction with a Qdot® streptavidin conjugate [4] (Figure 1).

Alexa Fluor® dye–labeled CT-B, a component of the Vybrant® Lipid Raft Labeling Kits, enables excellent endpoint and time-lapse imaging of lipid raft microdomains in the plasma membrane. However, when these probes are pushed to the limit—such as in time-lapse imaging with frequent, long, or intense exposures—photobleaching can occur (Figure 1A). Alternatively, when biotinylated CT-B was labeled with a Qdot® 655 streptavidin conjugate, the signal remained bright during similar acquisition conditions (Figure 1B).

Figure 1. Time-lapse imaging of lipid rafts in MMM murine macrophage cells. (A) Alexa Fluor® 594 dye–labeled cholera toxin subunit B (CT-B). (B) Qdot® 655 nanocrystal–labeled CT-B. Labeling experiments were performed at 4°C in complete medium. For Alexa Fluor® 594 labeling, we used the Vybrant® Alexa Fluor® 594 Lipid Raft Labeling Kit. For Qdot® 655 labeling, cells were incubated with 1 μg/mL biotinylated CT-B for 10 min followed by 10 nM Qdot® 655 streptavidin conjugate for 20 min and then a 1:200 dilution of anti–CT-B antibody (from the Vybrant® Lipid Raft Labeling Kit) for 15 min. Images were acquired at room temperature in Live Cell Imaging Solution. Alexa Fluor® 594 images were collected using 562/20 nm and 624/40 nm bandpass filters for excitation and emission, respectively. Qdot® 655 images were collected using a 435/20 nm bandpass excitation filter and a 515 nm longpass emission filter. Exposure times were adjusted to maximize the dynamic range of the fluorescent labeling.

Try Qdot® Conjugates in Place of Your Traditional Fluorophore Conjugates

Live-cell microscopy demands robust fluorophores that are bright, photostable, and multiplexable; these attributes are particularly important when imaging rare targets or highly dynamic cellular events such as membrane mobility and internalization processes. Qdot® nanocrystals meet these demands. Learn more about our wide selection of Qdot® conjugates and Qdot® labeling kits.

References

1. Pike LJ (2009) J Lipid Res S323–S328.
2. Simons K, Gerl MJ (2010) Nature Rev Mol Cell. Bio 11:688–699.
3. Lingwood D, Simons K (2010) Science 327:46–50.
4. Chakraborty SK, Bruchez MP, Ballou B et al. (2007) Nano Lett 7:2618-2626.