Light Sheet Fluorescence Microscopy (LSFM).

The development of confocal microscopy techniques introduced the ability to optically section fluorescent samples in the axial dimension, perpendicular to the image plane. These approaches, via the placement of a pinhole in the conjugate image plane, provided superior resolution in the axial (z) dimension resulting in nearly isotropic optical sections. However, increased axial resolution, via pinhole optics, comes at the cost of both speed and excitation efficiency. Light sheet fluorescent microscopy (LSFM), a century-old idea made possible with modern developments in both excitation and detection optics, provides sub-cellular resolution and optical sectioning capabilities without compromising speed or excitation efficiency. Over the past decade, several variations of LSFM have been implemented each with its own benefits and deficiencies. Here we discuss LSFM fundamentals and outline the basic principles of several major light-sheet-based imaging modalities (SPIM, inverted SPIM, multi-view SPIM, Bessel beam SPIM, and stimulated emission depletion SPIM) while considering their biological relevance in terms of intrusiveness, temporal resolution, and sample requirements.

Robust measurement of membrane bending moduli using light sheet fluorescence imaging of vesicle fluctuations.

The mechanical rigidity of lipid membranes is a key determinant of the energetics of cellular membrane deformation. Measurements of membrane bending moduli remain rare, however, and show a large variance, a situation that can be addressed by the development of improved techniques and by comparisons between disparate techniques applied to the same systems. We introduce here the use of selective plane illumination microscopy (SPIM, also known as light sheet fluorescence microscopy) to image thermal fluctuations of giant vesicles. The optical sectioning of SPIM enables high-speed fluorescence imaging of freely suspended vesicles and quantification of edge localization precision, yielding robust fluctuation spectra and rigidity estimates. For both lipid-only membranes and membranes bound by the intracellular trafficking protein Sar1p, which lowers membrane rigidity in a concentration-dependent manner, we show that the resulting bending modulus values are in close agreement with those derived from an independent assay based on membrane tether pulling. We also show that the fluctuation spectra of vesicles bound by the mammalian Sar1A protein, which stiffens membranes at high concentrations, are not well fit by a model of homogeneous quasi-spherical vesicles, suggesting that SPIM-based analysis can offer insights into spatially inhomogeneous properties induced by protein assemblies.

Modulation of membrane rigidity by the human vesicle trafficking proteins Sar1A and Sar1B.

The sculpting of membranes into highly curved vesicles is central to intracellular cargo trafficking, yet the mechanical activities of trafficking proteins remain poorly understood. Using an optical trap based assay that measures in vitro membrane response to imposed deformations, we examined the behavior of the two human paralogs of Sar1, a key component of the COPII family of vesicle coat proteins. Like their yeast counterpart, the human Sar1 proteins can lower the mechanical rigidity of the membranes to which they bind. Unlike the yeast Sar1, the rigidity is not a monotonically decreasing function of concentration. At high concentrations, we find increased bending rigidity and decreased protein mobility. These features imply a model in which protein clustering governs membrane mechanical properties.

The vesicle trafficking protein Sar1 lowers lipid membrane rigidity.

The sculpting of membranes into dynamic, curved shapes is central to intracellular cargo trafficking. Though the generation of membrane curvature during trafficking necessarily involves both lipids and membrane-associated proteins, current mechanistic views focus primarily on the formation of rigid cages and curved scaffolds by protein assemblies. Here we report on a different mechanism for the control of membrane deformation, unrelated to the imposition of predefined curvature, involving modulation of membrane material properties: Sar1, a GTPase that regulates vesicle trafficking from the endoplasmic reticulum, lowers the rigidity of the lipid bilayer membrane to which it binds. In vitro assays in which optically trapped microspheres create controlled membrane deformations revealed a monotonic decline in bending modulus as a function of Sar1 concentration, down to nearly zero rigidity, indicating a dramatic lowering of the energetic cost of curvature generation. This is the first demonstration that a vesicle trafficking protein lowers the rigidity of its target membrane, leading to a new conceptual framework for vesicle biogenesis.

Monolayer-protected nanoparticle film assemblies as platforms for controlling interfacial and adsorption properties in protein monolayer electrochemistry.

Assembled films of nonaqueous nanoparticles, known as monolayer-protected clusters (MPCs), are investigated as adsorption platforms in protein monolayer electrochemistry (PME), a strategy for studying the electron transfer (ET) of redox proteins. Modified electrodes featuring MPC films assembled with various linking methods, including both electrostatic and covalent mechanisms, are employed to immobilize cytochrome c (cyt c) for electrochemical analysis. The background signal (non-Faradaic current) of these systems is directly related to the structure and composition of the MPC films, including nanoparticle core size, protecting ligand properties, as well as the linking mechanism utilized during assembly. Dithiol-linked films of Au225(C6)75 are identified as optimal films for PME by sufficiently discriminating against detrimental background current and exhibiting interfacial properties that are readily engineered for cyt c adsorption and electroactivity (Faradaic current). Surface concentrations and denaturation rates of adsorbed cyt c are dictated by specific manipulation of the individual MPCs composing the outer layer of the film. The use of specially designed, hydrophilic MPCs as a terminal film layer results in near-ideal cyt c voltammetry, attributed to a high degree of molecular level control of the necessary interfacial interactions and flexibility needed to create a uniform and effective binding of protein across large areas of a substrate. The electrochemical properties of cyt c at MPC films, including ET rate constants that are unaffected by the large ET distance introduced by MPC assemblies, are compared to traditional strategies employing self-assembled monolayers to immobilize cyt c. The incorporation of nanoparticles as protein adsorption platforms has implications for biosensor engineering as well as fundamental biological ET studies.

Covalently networked monolayer-protected nanoparticle films.

Covalently networked films of nanoparticles can be assembled on various substrates from functionalized monolayer-protected clusters (MPCs) via ester coupling reactions. Exposure of a specifically modified substrate to alternating solutions of 11-mercaptoundecanoic acid exchanged and 11-mercaptoundecanol exchanged MPCs, in the presence of ester coupling reagents, 1,3-dicyclohexylcarbodiimide and 4-(dimethylamino)pyridine, results in the formation of a multilayer film with ester bridges between individual nanoparticles. These films can be grown in a controlled manner to various thicknesses and exhibit certain properties that are consistent with films having other types of interparticle connectivity, including chemical vapor response behavior and quantized double layer charging. Ester coupling of MPCs into assembled films is a straightforward and highly versatile approach that results in robust films that can endure harsher chemical environments than other types of films. The stability of these covalent films is assessed and compared to other more traditional MPC film assemblies.