A unique hyperspectral microscope (HSM) designed specifically to visualise molecular-level protein-protein interactions in living cells has been unveiled by the University of Albuquerque, designed around an Andor iXon 860 high-speed EMCCD detection system.
The novel design of the HSM provides acquisition rates of 27fps over a 28mm2 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 localisation with approximately 10nm precision even when the probes were clustered at spatial scales below the diffraction limit.
‘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,’ said Lidke. ‘Our imaging approach uses a spectrometer to spread light from 500nm to 750nm across 128 pixels of the camera. In our typical, high-speed configuration, we use half the camera and run at approximately 1,000fps, 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,’
According to Antoine Varagnat, product specialist at Andor: ‘The Andor iXon 860 EMCCD camera was able to meet the very challenging detection requirements of Professor Lidke's superfast hyperspectral microscope, namely ultra-low light detection at frame rates exceeding 1kHz. This level of performance was a key enabler for the team for the development of suitable tools for the study of the organisation and dynamics of their specific cellular components.
‘In their exciting paper, the capabilities of the new microscope were demonstrated by the application of high-resolution, spectrally-based particle tracking to observe membrane protein behaviour, including, for example, the dynamic formation and dissociation of epidermal growth factor receptor dimers, four-colour QD tracking while simultaneously visualising GFP-actin and high-density tracking for fast diffusion mapping.’
‘Many cellular signaling processes are initiated by dimerisation or oligomerisation of membrane proteins,’ said 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 multi-colour 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.’
