Imaging nanometre-scale structure in cells using in situ aberration correction.
Accurate distance measurements of cellular structures on a length scale relevant to single macromolecules or macromolecular complexes present a major challenge for biological microscopy. In addition to the inherent challenges of overcoming the limits imposed by the diffraction of light, cells themselves are a complex and poorly understood optical environment. We present an extension of the high-resolution colocalization method to measure three dimensional distances between diffraction-limited objects using standard widefield fluorescence microscopy. We use this method to demonstrate that in three dimensions, cells intrinsically introduce a large and variable amount of chromatic aberration into optical measurements. We present a means of correcting this aberration in situ [termed 'Colocalization and In-situ Correction of Aberration for Distance Analysis' (CICADA)] by exploiting the fact that there is a linear relationship between the degree of aberration between different wavelengths. By labelling a cellular structure with redundantly multi-colour labelled antibodies, we can create an intracellular fiducial marker for correcting the individual aberrations between two different wavelengths in the same cells. Our observations demonstrate that with suitable corrections, nanometre scale three-dimensional distance measurements can be used to probe the substructure of macromolecular complexes within cells.
Department of Biochemistry, Stanford University School of Medicine, Stanford, California, USA.
C J
CJ
Fuller
A F
AF
Straight
D004847:Epithelial Cells / Q000648:ultrastructure*; D006367:HeLa Cells; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods*; D046911:Macromolecular Substances / Q000648:ultrastructure*; D008853:Microscopy / Q000379:methods*
D046911:Macromolecular Substances