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.
Highlights
High-resolution live-cell imaging is necessary to study complex biological phenomena
The function of the structures is typically inferred from the presence of specific molecules of interest by performing targeted expression or immunolabelling, followed by fluorescence microscopy techniques resulting in correlative light and electron microscopy (CLEM)[7]
super-resolution optical fluctuation imaging (SOFI) increases the resolution of the widefield microscope by using the statistical information from stochastic intensity fluctuations and calculating the cumulants of the intensities between the individual pixels and image planes for resolution improvement in 2D and 3D respectively[11], (Fig. 1b)
Summary
High-resolution live-cell imaging is necessary to study complex biological phenomena. On the other hand, scanning probe microscopy (SPM) can obtain nanometer resolution images of the 3D surface of unlabeled living cells in their physiological environment[15] It has been proven as a versatile tool for biological imaging, and is often combined with fluorescence microscopy methods[16]. The most commonly used SPM technique, atomic-force microscopy (AFM), enables to study of the mechanobiology of fixed or living cells[17], to image biological membranes[18] and even track the dynamics of molecular assembly processes[19]. Label-free AFM methods combined with label-specific super-resolution fluorescence microscopy tools can provide maps of single-cells in unprecedented detail in fixed, and in living samples[20,21]. SICM is suitable for sensitive biological systems such as neurons, even with high-aspect ratio topographies with the invention of the hopping mode scanning modality[24]
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