Investigating Cellular Structures at the Nanoscale with Organic Fluorophores

Abstract

Super-resolution fluorescence imaging can provide insights into cellular structure and organization with a spatial resolution approaching virtually electron microscopy. Among all the different super-resolution methods single-molecule-based localization microscopy could play an exceptional role in the future because it can provide quantitative information, for example, the absolute number of biomolecules interacting in space and time. Here, small organic fluorophores are a decisive factor because they exhibit high fluorescence quantum yields and photostabilities, thus enabling their localization with nanometer precision. Besides past progress, problems with high-density and specific labeling, especially in living cells, and the lack of suited standards and long-term continuous imaging methods with minimal photodamage render the exploitation of the full potential of the method currently challenging.

Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

“…Chemical fluorophores, in comparison, are typically less than 1 kDa in size, and are brighter and more photostable. These properties allow chemical fluorophores to perform better than fluorescent proteins in advanced imaging modalities such as singlemolecule tracking and superresolution microscopies (3,4).…”

Localization microscopy coming of age: from concepts to biological impact

“…localization microscopy by dSTORM is feasible with many Alexa Fluor and ATTO dyes (van de Linde et al, 2011a;van de Linde et al, 2012;van de Linde et al, 2013;Wombacher et al, 2010;Klein et al, 2011;Jones et al, 2011). Post-translational labeling of proteins in living cells can be achieved using genetically encoded polypeptide tags in combination with organic fluorophore ligands (Miller and Cornish, 2005;Fernández-Suárez and Ting, 2008).…”

Imaging the invisible: resolving cellular microcompartments by superresolution microscopy techniques

“…By scanning the sample, images with a resolution of 40 m and below can be obtained. This technique has been very successfully applied for resolving protein organization in cellular microcompartments, such as dendritic spines (Nagerl et al , 2008 ) and synapses (Kittel et al , 2006 ) as well as plasma membrane microdomains (van den Bogaart et al , 2011 ). STED is in principle possible with every fluorescence dye but the imaging conditions have to be adjusted carefully to their photophysical properties.…”

“…In conventional STORM, photoactivation is achieved by energy transfer from another dye in close proximity (photochromic blinking), which provides flexible means for multicolor imaging (Bates et al , 2007 ). Meanwhile, reversible photoswitching in the absence of a sensitizing fluorophore as well as spontaneous return into the ground state has been demonstrated (direct STORM, dSTORM or ground state depletion microscopy followed by individual molecule return, GSDIM), thus simplifying the acquisition process substantially Heilemann et al , 2008 ;van de Linde et al , 2013 ). As a broad range of fluorophores is compatible with these approaches (Heilemann et al , 2008(Heilemann et al , , 2009Dempsey et al , 2011 ), multicolor superresolution imaging is readily implemented (Bates et al , 2007 ;Bossi et al , 2008 ;Testa et al , 2010 ).…”

“…By selectively targeting these dyes to proteins in live cells by posttranslational labeling techniques (Johnsson and Johnsson , 2007 ) dSTORM imaging of intracellular microcompartments is possible in living cells (Wombacher et al , 2010 ). Because of reversible photoswitching, dSTORM in living cells can be employed for capturing the dynamics of cellular nanostructures (Testa et al , 2010 ;Wombacher et al , 2010 ;Shim et al , 2012 ;Wilmes et al , 2012 ;van de Linde et al , 2013 ). (F)PALM and (d)STORM have very similar requirements in terms of laser illumination and detection capabilities of the microscope.…”