Z-range extension with complex PSF in 3D SMLM using Adaptive Optics

MicAO 3DSR – an all-in-one, versatile adaptive optics system: no need for a cylindrical lens or phase mask to optimize and shape the PSF in SMLM.

Photoactivation localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) enable localization of fluorescent molecules with nanometric resolution. In these super resolution microscopy methods the positions of molecules are obtained by fitting the shape of point spread function (PSF). Unfortunately, the PSF is symmetrical along Z axis, thus it does not provide the 3D information. In order to retrieve Z localization, the axial symmetry of the PSF has to be broken, typically using a PSF shaping technique.

The first and still the most popular PSF shaping method is to add astigmatism to the PSF using a cylindrical lens. This way the PSF extends in one direction above the focus and in the opposite direction below the focus, the amplitude of the extension being proportional to depth. The typical Z range provided by this method is reaching 1µm and localization precision up to 50nm. Practically, system and sample induced aberrations severely distort the PSF generated by a cylindrical lens, the amplitude of its extension being no longer symmetrical along Z. Using Adaptive Optics enables to compensate for aberrations, thus restoring Z localization precision, in particular in depth. Moreover, adaptive optics enables adding a controlled amount of perfect astigmatism, “cleaner” than by cylindrical lens. To this aim, our MicAO 3DSR add-on for SMLM microscopes is the perfect tool to easily optimize 3D localization precision and improve the axial resolution down to 20nm.

Sometimes 1µm axial range is not enough for observing the whole cellular structure in one acquisition process. Alternative PSF shaping methods can generate unique PSF shapes over an extended axial range. Among these methods, Tetrapod PSF – formed by a superposition of lower and higher order astigmatisms – provides an efficient combination of Z range extension, typically between 3 and 5µm, and localization precision. Tetrapod PSFs can be easily generated using a deformable mirror inside MicAO 3DSR. This capability makes MicAO 3DSR an extremely versatile tool to optimize PSF shaping in SLMLM, and to generate various PSF shapes depending on the need in terms of localization performance.

#AdaptiveOptics #Microscopy #3Dmicroscopy

Contact Imagine Optic