Bio-Imaging and Super-resolution

Imaging Intracellular Fluorescent Proteins at Nanometer Resolution

Eric Betzig,^1,2* George H. Patterson,³ Rachid Sougrat,³ O. Wolf Lindwasser,³ Scott Olenych,⁴Juan S. Bonifacino,³ Michael W. Davidson,⁴ Jennifer Lippincott-Schwartz,³ Harald F. Hess^5*

We introduce a method for optically imaging intracellular proteins at nanometer spatial resolution. Numerous sparse subsets of photoactivatable fluorescent protein molecules were activated, localized (to 2 to 25 nanometers), and then bleached. The aggregate position information from all subsets was then assembled into a superresolution image. We used this method—termed photoactivatedlocalization microscopy—to image specific target proteins in thin sections of lysosomes and mitochondria; in fixed whole cells, we imaged vinculin at focal adhesions, actin within a lamellipodium, and the distribution of the retroviral protein Gag at the plasma membrane.

Three-Dimensional Super-Resolution Imaging by Stochastic Optical Reconstruction Microscopy

Bo Huang,^1,2 Wenqin Wang,³ Mark Bates,⁴ Xiaowei Zhuang^1,2,3*

^{Recent advances in far-field fluorescence microscopy have led to substantial improvements in image resolution, achieving a near-molecular resolution of 20 to 30 nanometers in the two lateral dimensions. Three-dimensional (3D) nanoscale-resolution imaging, however, remains a challenge. We demonstrated 3D stochastic optical reconstruction microscopy (STORM) by using optical astigmatism to determine both axial and lateral positions of individual fluorophores with nanometer accuracy. Iterative, stochasticactivation of photoswitchable probes enables high-precision 3D localization of each probe, and thus the construction of a 3D image, without scanning the sample. Using this approach, we achieved an image resolution of 20 to 30 nanometers in the lateral dimensions and 50 to 60 nanometers in the axial dimension. This development allowed us to resolve the 3D morphology of nanoscopic cellular structures.}

Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure

Gleb Shtengel^a, 

James A. Galbraith^b, 

Catherine G. Galbraith^c, 

Jennifer Lippincott-Schwartz^d,¹,

Jennifer M. Gillette^d, 

Suliana Manley^d, 

Rachid Sougrat^d, 

Clare M. Waterman^e,

Pakorn Kanchanawong^e, 

Michael W. Davidson^f, 

Richard D. Fetter^a and 

Harald F. Hess^a,¹

Understanding molecular-scale architecture of cells requires determination of 3D locations of specific proteins with accuracy matching their nanometer-length scale. Existing electron and light microscopy techniques are limited either in molecular specificity or resolution. Here, we introduce interferometric photoactivated localization microscopy (iPALM), the combination of photoactivated localization microscopy with single-photon, simultaneous multiphase interferometry that provides sub-20-nm 3D protein localization with optimal molecular specificity. We demonstrate measurement of the 25-nm microtubule diameter, resolve the dorsal and ventral plasma membranes, and visualize the arrangement of integrin receptors within endoplasmic reticulum and adhesion complexes, 3D protein organization previously resolved only by electron microscopy. iPALM thus closes the gap between electron tomography and ight microscopy, enabling both molecular specification and resolution of cellular nanoarchitecture.

**Groupmeeting by Krister Shalm**