Cubic spline-based depth-dependent localization of mitochondria-endoplasmic reticulum contacts by three-dimensional...

The contact distance between mitochondria (Mito) and endoplasmic reticulum (ER) has received considerable attention owing to their crucial function in maintaining lipid and calcium homeostasis. Herein, cubic spline algorithm-based depth-dependent fluorescence-free three-dimensional light-sheet super-resolution microscopy (3D LSRM) with dual-wavelength illumination sources was investigated to study the distance of Mito-ER contacts in various live cells. To detect wavelength-dependent scattering, 12 nm gold nanoparticles (AuNPs) and 20 nm silver nanoparticles (AgNPs) as fluorescence-free nanoprobes were conjugated with Mito and ER. The cubic spline algorithm-based method showed improved localization precision in lateral and axial directions compared with that for previously used least squares and least cubic algorithms. The cubic spline-based depth-dependent localization was applied to the spatial localization of nanoprobes in super-resolution images, in which the average distance of Mito and ER was 22.4 nm in HeLa cells, 22.2 nm in RAW264.7 macrophage cells, 21.9 nm in AGS cells, 21.4 nm in HT29 cells, and 21.3 nm in HEK293 cells. The distances were ∼12% larger than those previously determined by electron microscopy, which demonstrated that this method was accessible and reliable for studying the intracellular structures of various live cells at the subdiffraction limit resolution.