A Simple Marker-Assisted 3D Nanometer Drift Correction Method for Superresolution Microscopy

Abstract

High-precision fluorescence microscopy such as superresolution imaging or single-particle tracking often requires an online drift correction method to maintain the stability of the three-dimensional (3D) position of the sample at a nanometer precision throughout the entire data acquisition process. Current online drift correction methods require modification of the existing two-dimensional (2D) fluorescence microscope with additional optics and detectors, which can be cumbersome and limit its use in many biological laboratories. Here we report a simple marker-assisted online drift correction method in which all 3D positions can be derived from fiducial markers on the coverslip of the sample on a standard 2D fluorescence microscope without additional optical components. We validate this method by tracking the long-term 3D stability of single-molecule localization microscopy at a precision of <2 and 5 nm in the lateral and axial dimension, respectively. We then provide three examples to evaluate the performance of the marker-assisted drift correction method. Finally, we give an example of a biological application of superresolution imaging of spatiotemporal alteration for a DNA replication structure with both low-abundance newly synthesized DNAs at the early onset of DNA synthesis and gradually condensed DNA structures during DNA replication. Using an isogenic breast cancer progression cell line model that recapitulates normal-like, precancerous, and tumorigenic stages, we characterize a distinction in the DNA replication process in normal, precancerous, and tumorigenic cells.

Correlative live and super-resolution imaging reveals the dynamic structure of replication domains

“…A recent report illustrates the possibility of implementing STORM in 3D for replication foci and reliably address the drift over time, making it an useful tool for setups equipped with optical sectioning (Ma et al, 2017).…”

Super-resolution imaging reveals the evolution of higher-order chromatin folding in early carcinogenesis

“…We then quantified the structural disruption of heterochromatin using two different methods -Gaussian mixed model clustering (Ma et al, 2017) and radial distribution function (RDF) (a.k.a. pair-correlation function) (Caetano et al, 2015;Xu et al, 2018).…”

“…The 60% of 2,2thiodiethanol (TDE, Sigma-Aldrich) in PBS was used to optically clear the tissue sections for ~20-30 minutes. The fluorescent beads (FluoSpheres TM carboxylate, F8803, Thermo Fisher Scientific) were added into the sample dish as fiduciary markers used for drift correction based on our previously published method (Ma et al, 2017).…”

Enhanced super-resolution microscopy by extreme value based emitter recovery

“…Following emitter recovery, we reconstructed the super-resolution images reconstructed by ThunderSTORM 13,14 , rendered as the 2D image (Figs. 3(b, d-e)) and the 3D scattered plot of localized emitter positions ( Fig.…”

“…The emitted fluorescence was collected by the objective, passing through the dichroic mirror (ZT488/640rpc-UF1, Chroma) and a band-pass emission filter (ZET488/640m, Chroma), and then focused by the tube lens and a 0.5X C-mount adapter onto a sCMOS camera (pco.edge 4.2, PCO-Tech), corresponding to a pixel size of 130 nm on the sample plane. A closed-loop piezo nanopositioner (Nano-F100S, Mad City Labs) was used for drift real-time correction tracking the 3D positions of fluorescence nanospheres (F8803, Thermo Fisher Scientific) 14 . Data acquisition, laser intensity control and drift correction were all integrated in our custom-designed software written in LabVIEW (National Instruments) and MATLAB (MathWorks).…”