Cubic B-spline calibration for 3D super-resolution measurements using astigmatic imaging.

Sven Proppert,Markus Sauer,Teresa Klein,Thorge Holm,Steve Wolter,Sebastian Van De Linde

doi:10.1364/oe.22.010304

Abstract

In recent years three-dimensional (3D) super-resolution fluorescence imaging by single-molecule localization (localization microscopy) has gained considerable interest because of its simple implementation and high optical resolution. Astigmatic and biplane imaging are experimentally simple methods to engineer a 3D-specific point spread function (PSF), but existing evaluation methods have proven problematic in practical application. Here we introduce the use of cubic B-splines to model the relationship of axial position and PSF width in the above mentioned approaches and compare the performance with existing methods. We show that cubic B-splines are the first method that can combine precision, accuracy and simplicity.

Highlights

Since the introduction of localization microscopy methods such as photoactivated localization microscopy (PALM) [1, 2], fluorescence photoactivation localization microscopy (FPALM) [3], stochastic optical reconstruction microscopy (STORM) [4,5] and direct STORM [6, 7], several techniques have been developed to enable precise 3D localization of single emitters
3D information is obtained by imaging the lateral plane and inferring the axial coordinate from the lateral width of the point spread function (PSF)
The natural PSF of a wide-field microscope yields little axial information due to two reasons: It is axially symmetric, and the PSF is insensitive to axial changes close to the focus

Summary

Introduction

Since the introduction of localization microscopy methods such as photoactivated localization microscopy (PALM) [1, 2], fluorescence photoactivation localization microscopy (FPALM) [3], stochastic optical reconstruction microscopy (STORM) [4,5] and direct STORM (dSTORM) [6, 7], several techniques have been developed to enable precise 3D localization of single emitters. The natural PSF of a wide-field microscope yields little axial information due to two reasons: It is axially symmetric, and the PSF is insensitive to axial changes close to the focus. For both reasons, the PSF has to be modified to contain axial information. With a modified PSF, resolutions of 15–20 nm laterally and 40–80 nm axially are achieved [8,9] (values expressed as full width at half maximum (FWHM)). Using interferometric PALM, axial resolutions of the order of 1–10 nm can be achieved, even though with restrictions to smaller axial ranges [10,11]

Methods

Results

Conclusion