Abstract
Multi-colour super-resolution localization microscopy is an efficient technique to study a variety of intracellular processes, including protein-protein interactions. This technique requires specific labels that display transition between fluorescent and non-fluorescent states under given conditions. For the most commonly used label types, photoactivatable fluorescent proteins and organic fluorophores, these conditions are different, making experiments that combine both labels difficult. Here, we demonstrate that changing the standard imaging buffer of thiols/oxygen scavenging system, used for organic fluorophores, to the commercial mounting medium Vectashield increased the number of photons emitted by the fluorescent protein mEos2 and enhanced the photoconversion rate between its green and red forms. In addition, the photophysical properties of organic fluorophores remained unaltered with respect to the standard imaging buffer. The use of Vectashield together with our optimized protocol for correction of sample drift and chromatic aberrations enabled us to perform two-colour 3D super-resolution imaging of the nucleolus and resolve its three compartments.
Highlights
Fluorescence microscopy has become an invaluable tool for cellular studies, both on structural and functional levels
The schemes are generally based on photoswitchable fluorescent proteins (PALM, photoactivation localization microscopy)[6,7] or organic dyes[8,9]
The nucleolus was chosen as a model system to apply this optimized protocol. This non enveloped nuclear compartment is subdivided into three sub-domains that are all associated with specific steps of ribosome biogenesis: fibrillar centres (FCs) – transcription of rDNA; dense fibrillar component (DFC) – pre-rRNA processing and modification; and granular component (GC) – assembly of ribosomal subunits[15]
Summary
Fluorescence microscopy has become an invaluable tool for cellular studies, both on structural and functional levels. The use of different fluorophores offers the possibility to visualize simultaneously different structures and to quantitatively characterize protein-protein interactions with the help of approaches such as Förster Resonance Energy Transfer coupled to Fluorescence Lifetime Imaging Microscopy (FRET-FLIM)[2] or Fluorescence Cross Correlation Spectroscopy (FCCS)[3] In their conventional implementations, these methods are restricted by the diffraction phenomenon that limits their spatial resolution to about 200 nm in the lateral plane and 500 nm along the axial direction. The nucleolus was chosen as a model system to apply this optimized protocol This non enveloped nuclear compartment is subdivided into three sub-domains that are all associated with specific steps of ribosome biogenesis: fibrillar centres (FCs) – transcription of rDNA; dense fibrillar component (DFC) – pre-rRNA processing and modification (methylation, pseudouridylation); and granular component (GC) – assembly of ribosomal subunits[15]. We applied a procedure to minimize and correct the chromatic aberrations inherent to any optical microscope in order to generate 2D and 3D images of the nucleoli
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