Abstract
Interferometric Photo-Activation-Localization-Microscopy (iPALM) localizes single fluorescent molecules with 20 nm lateral and 10 nm axial resolution. We present a method utilizing glass coverslip lithography for correlative imaging between iPALM and scanning electron microscopy (SEM). Using iPALM on HIV Gag-Dendra virus-like particles (VLPs) we localized the position of HIV Gag proteins. Based on these localizations we reconstructed the central cavity of the VLPs along with imperfections within the HIV Gag lattice. The SEM images and iPALM images overlap and show imaging from single VLPs immobilized on glass coverslips. The localization of many HIV proteins including accessory proteins and Gag-Pol remains unknown, we discuss how the specificity of iPALM coupled with SEM has the potential for resolving more of HIV proteins.
Highlights
Resolution of fluorescence microscopy is limited by diffraction; the advantage of fluorescence is that fluorescent tags are very specific
Once the general area of the Interferometric Photo-Activation-Localization-Microscopy (iPALM) imaging on the glass coverslip was identified with scanning electron microscopy (SEM), a detailed scan of 4 nm/pixel was commenced which covered the full area of the iPALM imaging
High resolution optical microscopy has been used to localize cellular components around HIV budding sites (Bleck et al 2014; Prescher et al 2015; Van Engelenburg et al 2014); kinetics of HIV virion release demonstrate a competition between HIV protease activation and virion release the mechanism of which remains unexplored and requires development of new imaging methodologies (Bendjennat and Saffarian 2016)
Summary
Resolution of fluorescence microscopy is limited by diffraction; the advantage of fluorescence is that fluorescent tags are very specific. The position of a single fluorescent molecule can be determined based on the center of its diffraction limited image with nanometer precision (Thompson et al 2002). It is possible to reconstruct an image by activating the molecules one at a time and obtaining their position with nanometer precision. This principle was explored in photoactivatable localization microscopy [PALM, (Kaksonen and Drubin 2006)] fluorescence photoactivatable localization microscopy [fPALM, (Hess et al 2006)] and stochastic optical reconstruction microscopy. Of Biology, University of Utah, Salt Lake City, USA [STORM, (Rust et al 2006)] to achieve in plane resolutions of 20 nm. The axial resolution of these techniques can be extended either through introduction of astigmatism associated with the out of plane images (Huang et al 2008) or using Biplane imaging (Juette et al 2008) both of which report an axial resolution of 50 nm and in focus resolution of 20 nm
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