FlAsH-PALM: Super-resolution Pointillist Imaging with FlAsH-Tetracysteine Labeling

Abstract

Super-resolution light microscopy including pointillist methods based on single molecule localization (e.g., PALM/STORM) allow to image protein structures much smaller than the diffraction limit (200-300 nm). However, commonly used labeling strategies such as antibodies or protein fusions have several important drawbacks, including the risk to alter the function or distribution of the imaged proteins. We recently demonstrated that pointillist imaging can be performed using the alternative labeling technique known as FlAsH, which better preserves protein function, is compatible with live cell imaging, and may help reach single nanometer resolution. We applied FlAsH-PALM to visualize HIV integrase in isolated virions or infected cells, allowing us to obtain sub-diffraction resolution images of this enzyme's spatial distribution and analyze HIV morphology without altering viral replication. The technique should also prove useful to image delicate proteins in intracellular vesicles and organelles at high resolution. Here, we present a detailed protocol in order to facilitate the application of FLAsH-PALM to other proteins and biological structures.

Early cytoplasmic uncoating is associated with infectivity of HIV-1

“…HIV-iGFP-tetracysteine-CA-iRFP670Vpr (VSV-G) viruses were labeled for 2 h in a rotation chamber at 21°C with TC-ReAsH II as described elsewhere (60,61), replacing β-mercaptoethanol incubation with DTT (1 mM).…”

Functional organization of cytoplasmic inclusion bodies in cells infected by respiratory syncytial virus

“…Just before imaging, an oxygen scavenger buffer, composed of glucose oxidase, catalase, glucose and MEA, was prepared (see ref. 58 ). In order to correct for lateral drift during image sequence acquisition and to register localizations from the two color channels, 100 nm fluorescent multicolor Tetraspec beads were added to the sample.…”

Fluorogenic probes for super-resolution microscopy

“…67 The achieved high precision allowed high resolution imaging of actin-rich structures, such as filopodia (FWHM = 45 nm). SiRhQ (35) performed well in multicolor localization microscopy as well, in combination with the fluorescent protein mEos2. SiRhO, 35 and mEos2 have sufficiently different activation cross-sections to allow successive imaging of the two colors (Fig.…”

“…33 To confirm this, Zimmer et al demonstrated FlAsH-based photoactivated localization microscopy (dubbed as FlAsH-PALM) with a spatial resolution ∼30 nm. 34,35 They studied Human Immunodefficiency Virus, HIV, by inserting the tetracysteine tag into integrase (IN), an enzyme that mediates the integration of viral DNA into the host genome.…”