Resolution Of Stimulated Emission Depletion Microscopy Research Articles

Investigation of the effects of polarisation and alignment method of retarders in isoSTED microscopy

Stimulated emission depletion (STED) microscopy is a powerful tool for observing subcellular structures beyond the diffraction-limited resolution. To achieve improved isotropic three-dimensional (3D) resolution in STED microscopy, 4Pi-geometry, which is equipped with two opposing objectives, is adopted; this approach is known as isoSTED microscopy. In isoSTED microscopy, constructive and destructive interference occur at the shared focal plane for lateral and axial depletion, respectively. The performance of isoSTED microscopy depends critically on the polarisation status and optical power differences of the two beams of the 4Pi cavity, which are adjusted using retarders. Misalignment of the retarders creates a nonzero core of the depletion focus owing to partial destructive interference. We analytically investigated the effects of retarder misalignments and proposed a simple and accurate alignment method for retarders. We also demonstrate that the proposed method can generate a completely destructive interference pattern and consequently achieve a sub-40-nm 3D resolution.

Theoretical study of laser intensity noise effect on CW-STED microscopy

Spatial resolution of stimulated emission depletion (STED) microscopy varies with sample labeling techniques and microscope components, e.g., lasers, lenses, and photodetectors. Fluctuations in the intensity of the depletion laser decrease achievable resolution in STED microscopy; the stronger the fluctuations, the higher the average intensity needed to achieve a given resolution. This phenomenon is encountered in every STED measurement. However, a theoretical framework that evaluates the effect of intensity fluctuations on spatial resolution is lacking. This paper presents an analytical formulation based on a stochastic model that characterizes the impact of the laser fluctuations and correlation time on the depletion efficiency in continuous-wave (CW) STED microscopy. We compared analytical results with simulations using a wide range of intensity noise conditions and found a high degree of agreement. The stochastic model used considers a colored noise distribution for the laser intensity fluctuations. Simple analytical expressions were obtained in the limit of small and large fluctuations’ correlation time. These expressions fitted very well the available experimental data. Finally, this work offers a starting point to model other laser noise effects in various microscopy implementations.

Sample Preparation for Multicolor STED Microscopy.

Stimulated emission depletion (STED) microscopy is one of the optical superresolution microscopy (SRM) techniques, more recently also referred to as nanoscopy, that have risen to popularity among biologists during the past decade. These techniques keep pushing the physical boundaries of optical resolution toward the molecular scale. Thereby, they enable biologists to image cellular and tissue structures at a level of almost molecular detail that was previously only achievable using electron microscopy. All the while, they retain the advantages of light microscopy, in particular with regards to sample preparation and flexibility of imaging. Commercially available SRM setups have become more and more available and also increasingly sophisticated, both in terms of optical performance and, importantly, ease of use. Institutional microscopy core facilities now offer widespread access to this type of systems. However, the field has grown so rapidly, and keeps growing, that biologists can be easily overwhelmed by the multitude of available techniques and approaches. From this vast array of SRM modalities, STED stands out in one respect: it is essentially an extension to an advanced confocal microscope. Most experienced users of confocal microscopy will find the transition to STED microscopy relatively easy as compared with some other SRM techniques. This also applies to STED sample preparation. Nonetheless, because resolution in STED microscopy does not only depend on the wavelength of the incident light and the numerical aperture of the objective, but crucially also on the square root of the intensity of the depletion laser and, in general, on the photochemical interaction of the fluorophore with the depletion laser, some additional considerations are necessary in STED sample preparation. Here we describe the single color staining of the somatostatin receptor subtype 2A (SSTR2A) and dual color staining of the trans-Golgi-network protein TGN 38 and the t-SNARE syntaxin-6 for STED in the endocrine cell line AtT20 and STED imaging of the samples, providing the protocols in as general a form as possible. The protocols in this chapter are used in this way in an institutional microscopy core facility.

The use of azimuthally polarized sinh-Gauss beam in STED microscopy

We examine the use of azimuthally polarized sinh-Gauss beams in stimulated emission depletion (STED) microscopy. Based on the vectorial diffraction theory, the exciting and depleting fields produced by azimuthally polarized sinh-Gauss beams with different beam parameters have been presented and analyzed. The results show that azimuthally polarized sinh-Gauss beam can produce a steeper depleting pattern, and an exciting pattern without side lobes. It is thus helpful to achieve super resolution in STED microscopy.