PSF shaping using adaptive optics for three-dimensional single-molecule super-resolution imaging and tracking

Abstract

We present a novel approach for three-dimensional localization of single molecules using adaptive optics. A 52-actuator deformable mirror is used to both correct aberrations and induce two-dimensional astigmatism in the point-spread-function. The dependence of the z-localization precision on the degree of astigmatism is discussed. We achieve a z-localization precision of 40 nm for fluorescent proteins and 20 nm for fluorescent dyes, over an axial depth of ~800 nm. We illustrate the capabilities of our approach for three-dimensional high-resolution microscopy with super-resolution images of actin filaments in fixed cells and single-molecule tracking of quantum-dot labeled transmembrane proteins in live HeLa cells.

Fluorescence nanoscopy. Methods and applications

“…Recent advances that address these problems include precompensation of aberrations through adaptive optics and PSF engineering [57,64,214], two-photon activation and/or excitation [58,59] and the application of light sheet microscopy concepts to PALM [60]. Red-shifted photoconvertible probes will also be demanded for in-depth imaging.…”

“…Several strategies have been put forward to achieve three-dimensional localisation microscopy: biplane detection [53], astigmatic imaging [54], engineered PSFs whose profiles unambiguously change with depth [55][56][57], confined two-photon activation [58][59][60], interferometric PALM (iPALM) [61] and highly inclined and laminated optical sheet illumination [62].…”

Explorer l’architecture du vivant à l’échelle moléculaire grâce à la microscopie de super-résolution PALM/STORM

“…L'image d'une molécule unique est maintenant un ellipsoïde dont la largeur selon les axes x et y est fonction de la distance au plan focal ( figure 3a). Une version plus élaborée consiste à remplacer la lentille cylindrique par un miroir déformable et à appliquer les méthodes d'optique adaptative pour modifier avec soin le front d'onde de la lumière émise [2]. Une deuxième approche consiste à imager la molécule simultané-ment dans deux plans légèrement séparés (de l'ordre de 400 nm), ce qui permet d'en inférer la position en z (figure 3b).…”

A Study of Dynamic Optical Tweezers Generation for Communication Networks

“…Dark-bright soliton control within a semiconductor add/drop multiplexer has shown promising applications [11][12]. when the high optical field is configured as an optical tweezer or potential well [13][14]. In applications, the term dynamics can be realized and is suitable for dynamic wells/tweezers control [15][16].…”