Dual-Objective Pointillism Microscopy Setup with Interferometric and Astigmatic Detection

Abstract

In recent years, under the general term of pointillism microscopy many techniques have been developed which employ sequential imaging of photoswitching fluorophores to circumvent the diffraction barrier in light microscopy (PALM, FPALM, STORM, dSTORM, etc.). Single molecules are localized in the plane perpendicular to the optical axis and several methods exist to obtain the axial coordinate, enabling three-dimensional imaging. The highest three-dimensional resolution in fluorescence microscopy can be obtained by combining pointillism microscopy with interferometric detection through two objectives (1). Here, the axial coordinate is acquired with great accuracy from intensity variations caused by interference. However, this approach is very demanding with respect to setup stability and sample preparation, as the coherence length of fluorescent molecules is in the range of 10 µm. Therefore already small changes in sample thickness and refractive index can prevent the coherent detection of a single fluorescent molecule. Another way to enhance localization accuracy by using two objectives is to employ this approach in combination with astigmatic detection, where the axial coordinate is calculated from astigmatism introduced by an additional cylindrical lens. This already yields a √2-fold increase of localization accuracy, as twice as many photons are collected (2), although the axial resolution of an interferometer is not reached. To make use of the advantages of both detection schemes, we built a setup offering interferometric as well as astigmatic detection. Movable mirrors allow choosing the detection pathway depending on the application and desired localization accuracy. We find that combining interferometric and astigmatic detection in one setup yields the most versatile instrument for studying a broad range of subcellular microcompartments. (1) G. Shtengel et al. Proc. Natl. Acad. Sci. U.S.A., 106(9):3125-3130, Mar 2009 (2) K. Xu et al. Nat. Methods, 9(2):185-188, Feb 2012