3D extended-range particle-localization microscopy using Airy-beam-based point-spread functions

[摘要] The precise localization of point emitters in optical microscopy has revolutionized our ability to super resolve subcellular structures beyond the diffraction limit and to study fundamental biological dynamics through single-molecule tracking. It also found its applications on a larger scale in blood-flow characterization, traction-force microscopy and microfluidic research including lab-on-chip experiments. However, traditional optical microscopy is typified by a restrictively small depth of field, i.e. the operable range in the axial direction is severely limited by diffraction to a thin layer in contrast to the relatively large field of view in the xy directions. This precludes the probing of three-dimensional (3D) behaviors of the sample. A number of techniques have emerged recently for 3D point localization, typically exploiting characteristic axial variations in the optical point-spread function (PSF) to deduce the axial coordinate of the emitter. However, existing approaches suffer from limitations such as small depth ranges, low optical throughput, complicated implementations or degraded performance in presence of overlapping PSFs.In this work, the lateral translation of the Airy beam is exploited to encode the emitters' axial coordinates and two novel approaches, namely the Airy-CKM (i.e. complementary kernel matching) and the twin-Airy techniques, are reported for 3D point-localization microscopy. The following advantages are clearly demonstrated: (1) The Airy-beam-based PSFs yield diffraction-free propagation, i.e. little diffractive spreading, which provides a greater axial range than existing techniques and makes them suitable for imaging thick samples. (2) Deconvolution-based localization algorithms are utilized, allowing for imaging of high emitter densities with overlapping PSFs. (3) The continuous phase profiles used to generate the Airy-beam-based PSFs can be implemented with refractive phase masks, which yield nearly 100% broadband optical throughput avoiding the photon inefficiency of spatial light modulators. (4) The simplicity in implementations (especially the twin-Airy technique) makes them applicable to most fluorescence microscopes. A statistical comparison to existing methods is performed by calculating their Cramer-Rao lower bounds, where the proposed approaches compare favorably to the state-of-the-art. The reported techniques are demonstrated to in vivo measurement of blood-flow dynamics in zebrafish embryos, which, to the best of the author's knowledge, is the first the application of pupil-engineered PSFs to hemodynamic measurements. In addition, time-resolved traction-force microscopy will be discussed briefly as an ongoing work.

[发布机构] University:University of Glasgow;Department:School of Physics and Astronomy

[关键词] 3D point localization microscopy, extended depth range, Airy-beam PSF, twin-Airy PSF, blood flow measurement, zebrafish, Cramer-Rao lower bounds.