Bewersdorf Laboratory
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SMS (single-molecule switching) microscopy (such as (F)PALM/(d)STORM/DNA-PAINT) circumvents the diffraction limit through precise localization of spatially well-separated fluorescent molecules. The sample is labeled with fluorescent probes which are sequentially switched on and off. After image acquisition, the single emitters are localized and the resulting position estimates are used to reconstruct an image with sub-diffraction resolution.
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In regular fluorescence microscopy, the fluorophores are excited by a laser beam at a wavelength blue-shifted relative to their emission spectrum (for example at 488 nm for a GFP-like spectrum). Typically, a few nanoseconds after excitation, the fluorophores relax spontaneously into their electronic ground states by emitting fluorescence (with the major emission contribution between 500 and 550 nm). FPALM relies on special photoactivatable fluorophores which will only fluoresce after having been activated by light at another wavelength (typically ~400 nm). Thus, fluorescence can be ‘switched on’ by the activation laser.
An FPALM image is produced in two steps.
STEP 2:
Image processing
STEP 1:
Image acquisition
The sample is illuminated homogeneously but at low intensity with light at the photoactivation wavelength to activate only a sparse subset of molecules at any time. These activated molecules are excited by light at the fluorescence excitation wavelength and the resulting fluorescence is detected with a sensitive CCD camera. The imaged molecules eventually bleach (or deactivate). Repeating this procedure for other molecules results in recording of a large fraction of the whole probe ensemble.
Each image contains diffraction limited images of only a few molecules, which are likely to be well separated. Analyzing the recorded images allows localization of separate molecules with a precision much better than the diffraction limit. Subsequent combination of the position estimates produces a sub-diffraction resolved image of the labeled structure.
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Currently, we are pushing our custom built SMS microscopes towards even better spatial resolution and faster 3D imaging of living cells. Visit our Publications page to see our latest results.