Does Super-Resolution Fluorescence Microscopy Obsolete Previous Microscopic Approaches to Protein Co-localization?

Abstract

Summary Conventional microscopy techniques, namely the confocal microscope or deconvolution processes, are resolution limited, ~250 nm, by the diffraction properties of light as developed by Ernst Abbe in 1873. This diffraction limit is appreciably above the size of most multi-protein complexes, which are typically 20–50 nm in diameter. In the mid 2000s, biophysicists moved beyond the diffraction barrier by structuring the illumination pattern and then applying mathematical principles and algorithms to allow a resolution of approximately 100 nm, sufficient to address protein subcellular colocalization questions. This “breaking” of the diffraction barrier, affording resolution beyond 200 nm is termed super resolution microscopy. More recent approaches include single molecule localization (such as PhotoActivated Localization Microscopy (PALM)/STochastic Optical Reconstruction Microscopy (STORM)) and point spread function engineering (such as STimulated Emission Depletion (STED) microscopy). In this review, we explain basic principles behind currently commercialized super resolution setups and address advantages and considerations in applying these techniques to protein colocalization in biological systems.

Dependence of descriptors of co-localization on microscope spatiotemporal resolution and the choice of regions of interest

“…Although there is an increasing awareness of the problems associated with the quantitative use of co‐localization data and of the need for automated and systematic image analysis (Bolte and Cordelieres, ; Comeau, Costantino, & Wiseman, ; Jaskolski, Mulle, & Manzoni, ; Oheim and Li, ), there are only a few studies that have addressed how the recent gains in spatial resolution affect co‐localization measurements (MacDonald, Baldini, & Storrie, ).…”

The cellular basis of platelet secretion: Emerging structure/function relationships

“…Despite a high instrument acquisition cost, ~$750,000, 3D–SIM should become a wide spread approach in platelet granule biology. A second new fluorescence microscopy approach, dSTORM, has even better XY resolution, <50 nm, and has also begun to be applied to the platelets [22]. This is sufficient resolution to map protein distributions within individual α-granules.…”

Stochastic optical reconstruction microscopy–based relative localization analysis (STORM-RLA) for quantitative nanoscale assessment of spatial protein organization

“…However, diffraction-limited imaging techniques are capable of achieving only 200- to 300-nm spatial lateral resolution and 500–700 nm in the z -axis (Nienhaus and Nienhaus, 2016). Thus what is assessed as colocalization using such techniques could reflect a range of possibilities, from proteins directly interacting via chemical bonds to proteins located within 300 nm of each other (Figure 1; MacDonald et al. , 2015).…”