Super-resolution microscopy (PALM)

A key technique for our team is the well-known PhotoActivated Localization Microscopy (PALM). With this imaging method, proposed in 2006 [1-3] we can characterize the phototransformable fluorescent proteins we develop at the single-molecule level, in conditions that are appropriate for further applications in biologically relevant projects. The principle of PALM is to circumvent the diffraction-limited optical resolution ( 200-300 nm) by photoactivating a few molecules at a time. Individual molecules in each acquired frame (typically with a time exposure of a few ms), can then be precisely superlocalized thanks to their spatial separation. The accumulation of thousands of such frames, each containing a small number of single molecules that are observed until photobleached then permits the reconstruction of a subdiffraction image, also called super-resolution image. Typically 20 nm resolution can be achieved in PALM in the imaging plane. Higher resolutions can be achieved in STORM (Stochastic Optical Reconstruction Microscopy) with appropriate organic dyes and a so-called « switching buffer », usually at the expense of the necessity to work with chemically fixed samples.
Our home-made setup is equipped with six CW solid-state lasers whose wavelengths are ranging from violet to red and intensities between 50-400 mW. The output of these lasers is tuned by a computer-controlled acousto-optical tunable filter (AOTF). Our motorized inverted microscope (Olympus IX81) is equipped with several objectives with magnification ranging from 5X to 100X. The illumination of the sample is quickly switchable between direct wide-field and total internal reflection fluorescence (TIRF) modes. Coupling to a spectrophotometer via an optical fiber is also possible for a better spectral characterization of a sample along with image acquisition. A special home-made detection system allows recording images with two EMCCD cameras (Photometrics Evolve 512), typically to achieve multicolor imaging. Our microscope is also equipped with astigmatism-based 3D super resolution capability.

The PALM microscope room and a zoom on the optical table with our setup.

[1] Betzig, E., et al., Imaging intracellular fluorescent proteins at nanometer resolution. Science, 2006. 313(5793): 1642-5
[2] Rust, M.J., et al., Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods, 2006. 3(10): 793-5
[3] Hess, S.T., et al., Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J, 2006. 91(11): 4258-72

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