Approaches toward super-resolution fluorescence imaging of mitochondrial proteins using PALM

Abstract

Mitochondria are difficult targets for microscopy because of their small size and highly compartmentalized, membranous interior. Super-resolution fluorescence microscopy methods have recently been developed that exceed the historical limits of optical imaging. Here we outline considerations and techniques in preparing to image the relative location of mitochondrial proteins using photoactivated localization microscopy (PALM). PALM and similar methods have the capacity to dramatically increase our ability to image proteins within mitochondria, and to expand our knowledge of the location of macromolecules beyond the current limits of immunoEM.

Fig. 1

A simulation of the relative space occupied by the diffraction of light from a single fluorescent protein. The white simulated point spread function as might be obtained from a fluorescence microscope is depicted in the center. The point source is represented as a black dot in the middle of the simulated PSF. This is overlaid onto an EM image of mitochondria from NIH3T3 cells. The lateral dimension of the simulated PSF is estimated from a 60X 1.45NA objective lens and is shown with the 200 nm scale bar.

Fig. 2

Various mitochondrial fusion protein strategies. A nonfunctional matrix highlighter using only the cleavable mitochondrial localization sequence fused in frame to dronpa is shown in (A). (B) Illustrates a functional TFAM protein fusion with the N-terminus of mEos2. A similar example for the helicase Twinkle is shown in (C) to emphasize the relative scale of fusion proteins. A C-terminal Dronpa fusion is shown in (D), which splits the mature protein sequence from an N-terminal mitochondrial localization sequence (LS). The relative fusion cassettes are to scale based on amino acid sequence length.

Fig. 3

PALM image from an LR-white section. Mitochondrial DNA nucleoid localized TFAM-mEos (red) and matrix localized COX8L-dronpa (green) imaged from a 100 nm thick section of cold polymerized LR-white. Localized molecules are rendered based on confidence of localization as described [5].