Super-resolution microscopy approaches for live cell imaging

Abstract

By delivering optical images with spatial resolutions below the diffraction limit, several super-resolution fluorescence microscopy techniques opened new opportunities to study biological structures with details approaching molecular structure sizes. They have now become methods of choice for imaging proteins and their nanoscale dynamic organizations in live cells. In this mini-review, we describe and compare the main far-field super-resolution approaches that allow studying endogenous or overexpressed proteins in live cells.

Figure 1

Schematic description of the superresolution microscopy approaches. All images for this didactic description are computer-generated. Object to be imaged consisted of fluorescent emitters (A) and corresponding diffraction-limited image (B). (C) In RESOLFT/STED, a focused excitation beam (cyan) superimposed with a doughnut-shaped depletion beam (red) are scanned over the sample to acquire an image at high resolution (down to ∼50–80 nm in live cells). (D) In SIM, after the required software reconstruction, multiple wide-field images are acquired using sinusoidal illumination grid patterns to obtain high-resolution images (down to ∼50–100 nm in live cells using nonlinear saturated illumination). (E) In single-molecule localization microscopy, a large number of wide-field images containing a few isolated single fluorescent emitters are successively acquired. A high-resolution image is reconstructed from the localizations of each individual molecule. Resolutions down to ∼50 nm are commonly achieved in live cells. In the example provided, we considered the detection of 80% of the molecules present in the object image. Scale bar represents 1 μm. To see this figure in color, go online.

Figure 2

Examples of achievements obtained with superresolution microscopy in live biological samples. (A) STED: continuous-wave STED images of the yellow fluorescent protein (citrine) targeted to the endoplasmic reticulum in live cells revealing small tubules (∼60 nm). Image sequences show morphing of the endoplasmic reticulum at arrows (pixel size = 20 nm, 10 s recording time per image). Scale bar = 1 μm. This figure was adapted from Hein et al. (6). (B) SIM: total-internal reflection microscopy image series of eGFP-α-tubulin in a live S2 cell and corresponding SIM images revealing the elongation followed by a rapid shrinking of a microtubule. Integration time of 270 ms per frame. This figure was adapted from Kner et al. (18). (C) PALM: numerous single trajectories of β3-integrin fused with mEOS2, obtained on a single MEF cell with PALM, revealing that β3-integrin undergo slower free-diffusion inside focal adhesions (gray) than outside, as well as confined diffusion and immobilization. Figure adapted from Rossier et al. (31). (D) STORM: spatial dynamics of cortical actin skeleton stained with Lifeact-HaloTag/ATTO655. Each reconstruction was obtained using 1000 frames (2 ms per frame). Scale bar = 1 μm. This figure was adapted from Wilmes et al. (47). (E) uPAINT: live cell superresolution imaging of membrane epidermal growth factor receptor (EGFR) dimers based on single-molecule fluorescence resonance energy transfer induced by fluorescent ligand activation. (Inset) Preferential cell-edge localization of EGFR dimers. In addition, uPAINT provides numerous single-molecule trajectories on a single cell, allowing the extraction of the diffusion properties of the EGFR dimer population from the whole-ligand-activated EGFR population. This figure was adapted from Winckler et al. (56). To see this figure in color, go online.