Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure

Abstract

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 light microscopy, enabling both molecular specification and resolution of cellular nanoarchitecture.fluorescence microscopy ͉ interferometry ͉ PALM ͉ photoactivated localization microscopy ͉ single molecule imaging A fundamental question in biomedical research is how specific, nanometer-scale biomolecules are organized into multicomponent micron-scale structural and signaling ensembles that facilitate cell function. For example, microtubules are built of 8-nm tubulin subunits that incorporate on the ultrastructural level into polymers 25 nm in diameter and Ͼ10 m in length that serve as the building blocks of superstructures such as mitotic spindles and flagella. However, key challenges remain for determining cellular ultrastructure with high molecular specificity. Because cellular structures are organized on the nanoscale, nanometer resolution is required. Immunoelectron microscopy (EM)-based approaches provide the necessary resolution, but they lack robust molecular specificity because the large size of the antibodies hampers their penetration into dense structures and the specificity of the antibody can be compromised by cross-reactivity and epitope masking caused by the harsh fixation often used for high-resolution EM. Fluorescence microscopy coupled with fluorescent protein (FP) fusion technology enables imaging cellular structure with exquisite molecular specificity, but the resolution of 3D images is diffraction-limited to Ϸ200 nm in the lateral and Ϸ500 nm in the axial direction, limiting conventional fluorescence to the characterization of cellular superstructure. Some of the recent fluorescence-based superresolution microscopy techniques (1-5) demonstrated a resolution of Ͻ100 nm in the vertical direction; however, this is still insufficient to bridge the resolution gap between cellular ultrastructure and superstructure. To achieve near-ultrastructural 3D resolution even for the limited photon outputs of highmolecular-specificity FPs, we have developed a single-photon multiphase interferometric scheme and integrated it with a lateral photoactivated localization microscopy (PALM) (6

Synthesis of a Far‐Red Photoactivatable Silicon‐Containing Rhodamine for Super‐Resolution Microscopy

“…A method to overcome the diffraction limit involves sequentially activating sparse subsets of fluorescent molecules, imaging them until they bleach, and localizing each molecule with high precision. This technique, called photoactivated localization microscopy (PALM) or direct stochastic optical reconstruction microscopy ( d STORM), enables imaging of cellular components at the nm scale1 and has been extended to three‐dimensions using interferometric methods2 and other strategies 3…”

“…Mitochondria do not contain actin but associate tightly with the cytoskeleton, providing an ideal test case. The cells were then imaged using iPALM under conditions optimized for mEos2 2. Compound 17 and mEos2 exhibited sufficiently different activation cross‐sections to allow successive imaging of the two colors.…”

Fluorescence nanoscopy. Methods and applications

“…Several strategies have been put forward to achieve three-dimensional localisation microscopy: biplane detection [53], astigmatic imaging [54], engineered PSFs whose profiles unambiguously change with depth [55][56][57], confined two-photon activation [58][59][60], interferometric PALM (iPALM) [61] and highly inclined and laminated optical sheet illumination [62].…”

“…Superb lateral and axial resolution (≈4 and 5.4 nm, respectively [32,33]) has been achieved sandwiching the sample between two objectives to exploit the self-interference of emitted photons while taking advantage of the extra budget of photons available for improved localisation in all directions (interferometric PALM, iPALM or 4Pi-LM) [32,33,61]. This approach is limited to a 1-μm-thick optical layer (although the sample can be thicker) and is constrained by the severe complexity of the experimental setup.…”

Correlative iPALM and Platinum Replica Electron Tomography to Highlight Single Molecules on Clathrin Endocytic Structures in 3D

“…However, identifying proteins of interest in these images with immunogold requires large metal particles (15 nm) that not only label sparsely but also obstruct the image beneath. iPALM localizes fluorescently labeled proteins to a precision of better than 20 nm in the membrane plane (XY) and 10 nm in the axial (Z) plane [4]. Our iPALM/PREM correlation method allows us to correlate clathrin fluorescence with clathrin viewed with EM, to within 20 nm across a 20 µm cell membrane.…”