Correlative 3D microscopy of single cells using super-resolution and scanning ion-conductance microscopy

Abstract

High-resolution live-cell imaging is necessary to study complex biological phenomena. Modern fluorescence microscopy methods are increasingly combined with complementary, label-free techniques to put the fluorescence information into the cellular context. The most common high-resolution imaging approaches used in combination with fluorescence imaging are electron microscopy and atomic-force microscopy (AFM), originally developed for solid-state material characterization. AFM routinely resolves atomic steps, however on soft biological samples, the forces between the tip and the sample deform the fragile membrane, thereby distorting the otherwise high axial resolution of the technique. Here we present scanning ion-conductance microscopy (SICM) as an alternative approach for topographical imaging of soft biological samples, preserving high axial resolution on cells. SICM is complemented with live-cell compatible super-resolution optical fluctuation imaging (SOFI). To demonstrate the capabilities of our method we show correlative 3D cellular maps with SOFI implementation in both 2D and 3D with self-blinking dyes for two-color high-order SOFI imaging. Finally, we employ correlative SICM/SOFI microscopy for visualizing actin dynamics in live COS-7 cells with subdiffraction-resolution.

Time-Resolved Scanning Ion Conductance Microscopy for Three-Dimensional Tracking of Nanoscale Cell Surface Dynamics

“…The combination of SICM with high-performance optical microscopy is a very promising area . The SICM is integrated with an inverted optical microscope (Figure S23) and can thus be used with many of the recently developed super-resolution microscopy techniques …”

“…33 The SICM is integrated with an inverted optical microscope (Figure S23) and can thus be used with many of the recently developed super-resolution microscopy techniques. 34 The SICM field has developed rapidly in recent years, with many additional measurement capabilities being added, such as sample stiffness, 35 surface charge, 32 and local pH. 36 Combining these developments with time-resolved SICM or high-speed SICM will provide insights into eukaryotic membrane processes with three-dimensional nanometer detail.…”

A OneStep Solution to Fix and Stain Cells for Correlative Live and Fixed Microscopy

“…For example, light microscopy can be used to observe the behavior of virtually any cell type, while electron microscopy can reveal subcellular structures at much higher resolution in fixed cells. To take advantage of more than one type of microscopy at a time, cell biologists have developed several techniques to correlate data from multiple imaging modalities (Fernandes, Saavedra‐Villanueva, Margeat, Milhiet, & Costa, 2020; Gómez‐Varela et al., 2020; Navikas et al., 2021; Phillips et al., 2020). For example, Correlative Light and Electron Microscopy (CLEM) allows direct association of light microscopy data (e.g., fluorescent protein localization) with higher‐resolution electron microscopy (reviewed in de Boer, Hoogenboom, & Giepmans, 2015).…”

Data-driven microscopy allows for automated targeted acquisition of relevant data with higher fidelity

“…Because of the inherent spatial resolution of this domain, such systems are difficult to calibrate and force researchers into using single-system multi-modal imaging (1, 2), with on-stage fixation and labeling protocols (1, 3, 4). On the other hand, multi-system correlative imaging (5, 6, 7, 8, 9) is dependent on finding a transformation between the respective coordinate systems. While differences between the systems may prohibit off-the-shelf registration solutions (10, 11, 12, 13), the introduction of external cues such as markings (6) or calibration beads may influence the sample and obstruct image quality.…”

“…On the other hand, multi-system correlative imaging (5, 6, 7, 8, 9) is dependent on finding a transformation between the respective coordinate systems. While differences between the systems may prohibit off-the-shelf registration solutions (10, 11, 12, 13), the introduction of external cues such as markings (6) or calibration beads may influence the sample and obstruct image quality. Ideally, reference points should be derived from as little information as possible to achieve multi-system correlative imaging consistently.…”