Multi-color quantum dot tracking using a high-speed hyperspectral line-scanning microscope

• Published in: PloS one Vol. 8; no. 5; p. e64320
• Main Authors: Cutler, Patrick J, Malik, Michael D, Liu, Sheng, Byars, Jason M, Lidke, Diane S, Lidke, Keith A
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 22.05.2013; Public Library of Science (PLoS)
• Subjects: Actin; Astronomy; Biology; Cameras; Cell Line, Tumor; Color; Diffraction; Diffusion rate; Dimerization; Dimers; Dissociation; Emission spectra; Engineering; Epidermal growth factor; Frames per second; High speed; Humans; Immunoglobulin E; Laboratories; Light; Light diffraction; Localization; Medical research; Membrane proteins; Membrane Proteins - metabolism; Microscopy; Microscopy - instrumentation; Microscopy - methods; Oligomerization; Oligomers; Particle tracking; Physics; Proteins; Quantum Dots; Reproducibility of Results; Signal Transduction; Signaling; New Mexico; United States > US
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0064320

Many cellular signaling processes are initiated by dimerization or oligomerization of membrane proteins. However, since the spatial scale of these interactions is below the diffraction limit of the light microscope, the dynamics of these interactions have been difficult to study on living cells. We have developed a novel high-speed hyperspectral microscope (HSM) to perform single particle tracking of up to 8 spectrally distinct species of quantum dots (QDs) at 27 frames per second. The distinct emission spectra of the QDs allows localization with ∼10 nm precision even when the probes are clustered at spatial scales below the diffraction limit. The capabilities of the HSM are demonstrated here by application of multi-color single particle tracking to observe membrane protein behavior, including: 1) dynamic formation and dissociation of Epidermal Growth Factor Receptor dimers; 2) resolving antigen induced aggregation of the high affinity IgE receptor, FcεR1; 3) four color QD tracking while simultaneously visualizing GFP-actin; and 4) high-density tracking for fast diffusion mapping.