3D multicolor PALM/STORM
The resolution limit to define two very near molecules is defined by the Rayleigh distance (0,61λ/NA). With the classic microscopy we cannot distinguish two molecules that are at a distance inferior to that Rayleigh distance.
The single molecule microscopy allows the detection of a single molecule labeled with a fluorophore. Its exact position is most often determined by fitting its intensity profile with a gaussian function. The localization precision depends mainly on the number of the detected photons. This technique allows to localize the position of the molecules with a precision in the order of 10-20 nm, an order of magnitude smaller than the resolution limit. However, if more molecules are found at the same diffraction Airy pattern, the super-localization of their position is not possible.
The PALM/STORM techniques are based on the principle similar to pointillism. For every acquisition, a part of the fluorescent molecules is used, in order to distinguish and then localise every emitting molecule. The accumulation of the acquisition permits the reconstruction of a super-resolved image (figure 1).
PALM : PhotoActivated Localization Microscopy
The development of new fluorescent probes paved the way towards new strategies of the fluorescence microscopy. The use of photoactivable and photoconvertible proteins allowed the development of PALM microscopy (PA-GFP, PA-tagRFP, PA-mCherry, mEos2, Dendra2, etc). It is a technique based on the detection of single molecules. With the help of ultraviolet light (405nm), the photo-activable molecules will become fluorescent or the photoconvertible molecules will shift their emission spectrum. Using this type of molecules with 2 laser beams (one for the photoactivation or photoconversion and the other for the read-out), we excite every time a small part of the molecules, we detect them (=localize them with a localization precision one order of magnitude smaller to the resolution limit), we photobleach them and then we excite and detect the others, who will be photoactivated or photoconverted with the UV laser. The final image is consisted of the sum of all detected images.
The technique can be used for fixed samples. For living cells, the single molecule tracking is possible, even in dense samples (we just need to photoactivate the right amount of molecules every time so not to have an overlap of the Airy patterns), and then the dynamics of the molecules can be studied.
STORM : STochastic Optical Reconstruction Microscopy
The organic probes (pair Cy3-Cy5) are used for STORM microscopy. The dSTORM (direct STORM) is a technique that uses one or more organic labels and a buffer with oxido-redaction properties. With this buffer and a UV laser we can control the photodynamics of the molecule, which enables us to visualize every time a low density of the molecules, to have at the end the super-localization of each molecule and a super-resolved image.
The organic labels are coupled with an antibody, so cells are impermeabilized. The choice for living cells experiments is still limited. However, the signal is much stronger than the one with fluorescent proteins, so the localization precision is higher.
The facility disposes an ELYRA PS.1 Zeiss microscope. There is a TIRF excitation mode available. The ZEN software disposes a real time mode to visualize the super-resolved image live. The final super-resolved image can also be calculated by the software, with the possibility of adjusting various filtering parameters.
The microscope disposes a 3D function, for super-resolution images along 1.5μm.
Objectives : Immersion Zeiss 100x , NA 1.46
Immersion Zeiss 100x, NA 1.56
Immersion Zeiss 63x, NA 1.40
Excitation Lasers : 405 nm (50mW), 488 nm (300mW), 561 nm (200mW), 641 nm (150mW).
EMCCD Andor iXon 897 (512x512, pixel size 16μm, QE=90%).
The microscope disposes a 3D fonction, that allows a z range PALM/STORM imaging of 1.5μm.