Abstract
Due to low labeling efficiency and structural heterogeneity in fluorescence-based single-molecule localization microscopy (SMLM), image alignment and quantitative analysis is often required to make accurate conclusions on the spatial relationships between proteins. Cryo-electron microscopy (EM) image alignment procedures have been applied to average structures taken with super-resolution microscopy. However, unlike cryo-EM, the much larger cellular structures analyzed by super-resolution microscopy are often heterogeneous, resulting in misalignment. And the light-microscopy image library is much smaller, which makes classification challenging. To overcome these two challenges, we developed a method to deform semi-flexible ring-shaped structures and then align the 3D structures without classification. These algorithms can register semi-flexible structures with an accuracy of several nanometers in short computation time and with greatly reduced memory requirements. We demonstrated our methods by aligning experimental Stochastic Optical Reconstruction Microscopy (STORM) images of ciliary distal appendages and simulated structures. Symmetries, dimensions, and locations of protein complexes in 3D are revealed by the alignment and averaging for heterogeneous, tilted, and under-labeled structures.
Highlights
In the past decade, the development of localization-based super-resolution microscopy has brought the light microscopy to nanometer scales
As characterized by Fourier Ring Correlation (FRC) analysis, we have shown that our deformed alignment algorithm achieved better aligned image resolution for semi-flexible structures compared to the state-of-the-art rigid registration algorithm [12]
The deformation improves the resolution and symmetry in the aligning result. We demonstrated this improvement, by comparing the average image of structures aligned by the deformed alignment algorithm (Fig 1H) to the aligning result of the rigid registration algorithm recently developed in our group [12] (Fig 1J)
Summary
The development of localization-based super-resolution microscopy has brought the light microscopy to nanometer scales. Imaging beyond the diffraction limit addressed structural and functional biomedical questions of subcellular organelles that could not be resolved by conventional light microcopy. The in situ dissection of macromolecular protein complexes includes ciliary transition zone [1,2,3], neuronal synapses [4], nuclear pore complex [5, 6], focal adhesion complex [7], clathrin-coated pits [8], centrosome [9, 10], and the escort complex at viral budding sites [11]. To obtain optical super-resolution images, the target proteins or DNA/RNA sequences are fluorescently labeled by organic dyes or fluorescent protein tags.
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