University of Albuquerque Develops High-speed Hyperspectral Microscope
- Conceptual diagram of 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 coloured spheres denote those which are excited. The emitted light passes through the dichroic mirror and into a spectrometer, which distributes the light onto the Andor iXon 860 high-speed 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 backprojected 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 fps, providing spatial, spectral, and temporal resolution that enables localized single molecule tracking of multiple emitters within a given diffraction limited volume.
The University of Albuquerque has designed a hyperspectral microscope (HSM) around an Andor iXon 860 high-speed EMCCD detection system to visualize membrane receptor dynamics at the molecular level in living cells.
The HSM provides acquisition rates of 27 fps over a 28 square micrometer 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 Professor Keith Lidke, the New Mexico team performed single particle tracking of up to eight 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.
Statement of Professor Keith Lidke
"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 in living cells. Our unique, high-speed HSM enables multi-color single particle tracking of up to eight different probes simultaneously and has allowed us to directly observe the behavior of small signaling complexes that cannot be resolved with other diffraction-limited light microscopy techniques."
"We chose the Andor iXon 860 EMCCD camera to capture our signals because this demanding application involving high-speed acquisition under very low light conditions places real demands on detector technology to perform at significantly higher levels of sensitivity and speed. Our imaging approach uses a spectrometer to spread light from 500 nm to 750 nm across 128 pixels of the camera. In our typical, high-speed configuration, we use half the camera and run at approx. 1,000 fps with most pixels collecting just a few photons per frame. Electron Multiplying CCD (EMCCD) technology, as seen in the Andor iXon camera, amplifies down to single photons and is ideal for these studies."