Super-resolution Microscopy of Lipid Bilayer Phases

Abstract

Sub-diffraction optical imaging with nanometer resolution of lipid phase separated regions is reported. Merocyanine 540, a probe whose fluorescence is sensitive to the lipid phase, is combined with super-resolution imaging to distinguish the liquid and gel phase nanoscale domains of lipid bilayers supported on glass. The monomer-dimer equilibrium of MC 540 in membranes is deemed responsible for the population difference of single molecule fluorescence bursts in the different phase regions. The extension of this method to other binary or ternary lipid models or natural systems provides a promising new super-resolution strategy.

Analytical use of multi-protein fluorescence resonance energy transfer to demonstrate membrane-facilitated interactions within cytokine receptor complexes

“…When they are isolated in vitro from cells, they are defined as detergent-resistant membranes. Lipid rafts have distinct biophysical properties (e.g., increased viscosity) and likely form a distinct physical phase in the membrane from those of aggregates formed by other lipid molecules [11,[24][25][26][27][28][29][30]. Lipid rafts are likely to be quasi-stable -although these lipid rafts are physically separated from other sections of the plasma membrane on a low-nanometer scale, individual rafts are not clearly resolved using visible-light optics nor are they stationary on a high-nanometer scale.…”

Three-dimensional superresolution colocalization of intracellular protein superstructures and the cell surface in live Caulobacter crescentus

“…One class, based on spatial patterning of the excitation and/or a nonlinear response, includes three-dimensional (3D) structured illumination microscopy (3), which improves resolution by a factor of two, and 3D stimulated emission depletion microscopy (4), which has been demonstrated to have an isotropic resolution of approximately 40 nm depending upon the emitter. The complementary class, denoted by the mechanism-independent term single-molecule active control microscopy (SMACM), includes (fluorescence) photoactivated localization microscopy [(F) PALM] (5, 6), [direct (7)] stochastic optical reconstruction microscopy (STORM) (8), points accumulation for imaging in nanoscale topography (PAINT) (9,10), photoswitching of fluorescent proteins (11), ground state depletion microscopy followed by individual molecule return (12), and blink microscopy (13). All of these pointillist techniques localize sparse subsets of singlemolecule emitters sequentially over time, thereby building up a superresolution reconstruction, until the desired structure is resolved.…”

“…Here, we describe a method for sequential localization of an internal protein superstructure and the bacterial surface, termed superresolution by power-dependent active intermittency and points accumulation for imaging in nanoscale topography (SPRAIPAINT), which is a combination of photo-induced blinking of eYFP (SPRAI) and transient labeling of the cell surface via PAINT (9,10). This method features the advantages of requiring a single reading laser, and PAINT can be performed indefinitely because of the continuous replenishment of dye from solution.…”

3D Super-Resolution Imaging with Blinking Quantum Dots

“…Due to advantages of quantum dots over organic fluorophores, the new technique QDB3 could be a better choice in certain situations than other three dimensional super-resolution imaging techniques 21–29,50,51 . Generally speaking, for example, higher precision of localization can be achieved because quantum dots are intensely bright, compared to most organic fluorophores (roughly localization precision $\propto 1/\sqrt{N}$ where N is the collected photon number) 52,53 .…”