Nanoscopy in a Living Multicellular Organism Expressing GFP

Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Biophysical Journal (Impact Factor: 3.97). 06/2011; 100(12):L63-5. DOI: 10.1016/j.bpj.2011.05.020
Source: PubMed


We report superresolution fluorescence microscopy in an intact living organism, namely Caenorhabditis elegans nematodes expressing green fluorescent protein (GFP)-fusion proteins. We also superresolve, by stimulated emission depletion (STED) microscopy, living cultured cells, demonstrating that STED microscopy with GFP can be widely applied. STED with GFP can be performed with both pulsed and continuous-wave lasers spanning a wide wavelength range from at least 556-592 nm. Acquiring subdiffraction resolution images within seconds enables the recording of movies revealing structural dynamics. These results demonstrate that numerous microscopy studies of live samples employing GFP as the marker can be performed at subdiffraction resolution.

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Available from: Daniel A Colón-Ramos
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    • "In addition, in order to corroborate that our results were not dependent on the sample preparation or imaging technique, we directly measured GFP-Bax distribution (without staining with nanobodies) during apoptosis with an alternative super-resolution method, stimulated emission depletion microscopy (STED). This technique has also showed improvements in resolution up to 60 nm for imaging of GFP-labeled molecules (Willig et al, 2006;Rankin et al, 2011). As observed in Fig 4C,the images obtained with STED showed a substantial resolution increase compared to those acquired with diffraction-limited imaging. "
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    • "Further, the use of a continuouswave laser in conjunction with a gated fluorescence detection scheme has enabled a reduction in the STED laser power and a simplification of the setups (Vicidomini et al. 2011; Honigmann et al. 2012a; Mueller et al. 2012). As a consequence , STED microscopy is nowadays considered a straightforward technique for the study of the living cell using genetically encoded markers such as fluorescent proteins (Willig et al. 2006a; Hein et al. 2008; Nagerl et al. 2008; Eggeling et al. 2009; Li et al. 2009; Moneron and Hell 2009; Morozova et al. 2010; Rankin et al. 2011; Tonnesen et al. 2011; Urban et al. 2011), tagging proteins such as SNAP-, HALO-, or CLIP-tags (Schr€ oder et al. 2008; Eggeling et al. 2009; Hein et al. 2010; Pellett et al. 2011; Lukinavicius et al. 2012), or fluorogen-activating tags (Fitzpatrick et al. 2009) (which both covalently bind functionalized and membrane-permeable organic dyes), even using commercial instrumentation (Schr€ oder et al. 2008; Fitzpatrick et al. 2009; Morozova et al. 2010; Friedemann et al. 2011). Furthermore, the quite high laser intensities of 1– 10 MW/cm 2 can be circumvented when switching from STED to RESOLFT nanoscopy, which employs laser intensities in the range of 1–5 kW/cm 2 only (Hofmann et al. 2005; Brakemann et al. 2011; Grotjohann et al. 2011; Testa et al. 2012). "
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    ABSTRACT: Recent developments in fluorescence far-field microscopy such as STED microscopy have accomplished observation of the living cell with a spatial resolution far below the diffraction limit. Here we briefly review the current approaches to super-resolution optical microscopy and present the implementation of STED microscopy for novel insights into live cell mechanisms, with a focus on neurobiology and plasma membrane dynamics. © 2013 International Society for Neurochemistry, J. Neurochem. (2013) 10.1111/jnc.12243.
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    • "This apparent discrepancy is explained by the resolution limit of conventional light microscopy: two closely located 300 nm long invaginations can be mistaken for one when imaged using fluorescence microscopy (Stradalova et al. 2009). Accordingly, the number of eisosomes per cell seen by super-resolution microscopy is higher than previously calculated (Rankin et al. 2011). Thus, a Pil1-GFP (or Sur7-GFP) plasma membrane fluorescent punctum should not be interpreted as a single eisosome. "
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