Driven by an electron-multiplying charge-coupled device (EMCCD) detection system (Andor iXon 860), the HSM's design delivers 27 frame/s acquisition rates over a 28 µm2 field of view with each pixel collecting 128 spectral channels, allowing the determination of stoichiometry and dynamics of small oligomers unmeasurable by any other technique. Led by Prof. Keith Lidke, an assistant professor in the Department of Physics & Astronomy at the university, the research team performed single-particle tracking of up to 8 spectrally distinct species of quantum dots (QDs), the distinct emission spectra of the QDs allowing localization with approx. 10 nm precision, even when the probes were clustered at spatial scales below the diffraction limit.
|Conceptual diagram of the high-speed hyperspectral microscope: The excitation beam is reflected by a dichroic mirror and forms a laser line focused at the sample plane by the objective, concentrating the excitation light to a small volume of diffraction-limited width. The white spheres in the sample represent fluorophores that remain mostly in the ground state, while the colored spheres denote those which are excited. The emitted light passes through the dichroic mirror and into a spectrometer, which distributes the light onto the EMCCD camera such that each exposure captures information of wavelength and position along the line. The entrance slit on the spectrometer also serves to reject out-of-focus light, providing a semi-confocal ability for imaging at any depth in the sample. A scanning mirror (not shown) advances the line position by one back-projected pixel length on the sample and another exposure is acquired. One hyperspectral ‘‘frame’’ is a reconstructed series of these steps (performed in post-processing) to form an image containing x, y, and l. A time series of these hyperspectral frames is acquired at 27 frames/s, providing spatial, spectral, and temporal resolution that enables localized single-molecule tracking of multiple emitters within a given diffraction-limited volume.|
"Many cellular signaling processes are initiated by dimerization or oligomerization of membrane proteins," says Lidke. "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 in living cells. Our unique, high-speed HSM enables multicolor single particle tracking of up to eight different probes simultaneously and has allowed us to directly observe the behaviour of small signaling complexes that cannot be resolved with other diffraction-limited light microscopy techniques."
Lidke adds that the approach uses a spectrometer to spread light from 500 to 750 nm across 128 pixels of the camera. In the team's typical, high-speed configuration, they use half the camera and run at approx. 1000 frames/s, with most pixels collecting just a few photons per frame. EMCCD technology enables amplification down to single photons, which is ideal for their work, he explains.
Full details of the work appear in the journal PLoS One; for more information, please visit http://dx.doi.org/10.1371/journal.pone.0064320.
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