Samuel T. Hess

Imaging coexisting fluid domains in biomembrane models coupling curvature and line tension

Lipid bilayer membranes—ubiquitous in biological systems and closely associated with cell function—exhibit rich shape-transition behaviour, including bud formation and vesicle fission. Membranes formed from multiple lipid components can laterally separate into coexisting liquid phases, or domains, with distinct compositions. This process, which may resemble raft formation in cell membranes, has been directly observed in giant unilamellar vesicles. Detailed theoretical frameworks link the elasticity of domains and their boundary properties to the shape adopted by membranes and the formation of particular domain patterns, but it has been difficult to experimentally probe and validate these theories. Here we show that high-resolution fluorescence imaging using two dyes preferentially labelling different fluid phases directly provides a correlation between domain composition and local membrane curvature. Using freely suspended membranes of giant unilamellar vesicles, we are able to optically resolve curvature and line tension interactions of circular, stripe and ring domains. We observe long-range domain ordering in the form of locally parallel stripes and hexagonal arrays of circular domains, curvature-dependent domain sorting, and membrane fission into separate vesicles at domain boundaries. By analysing our observations using available membrane theory, we are able to provide experimental estimates of boundary tension between fluid bilayer domains.

Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples

Imaging volumes as thick as whole cells at three-dimensional (3D) super-resolution is required to reveal unknown features of cellular organization. We report a light microscope that generates images with translationally invariant 30 × 30 × 75nm resolution over a depth of several micrometers. This method, named biplane (BP) FPALM, combines a double-plane detection scheme with fluorescence photoactivation localization microscopy (FPALM) enabling 3D sub-diffraction resolution without compromising speed or sensitivity.

Large-scale fluid/fluid phase separation of proteins and lipids in giant plasma membrane vesicles

The membrane raft hypothesis postulates the existence of lipid bilayer membrane heterogeneities, or domains, supposed to be important for cellular function, including lateral sorting, signaling, and trafficking. Characterization of membrane lipid heterogeneities in live cells has been challenging in part because inhomogeneity has not usually been definable by optical microscopy. Model membrane systems, including giant unilamellar vesicles, allow optical fluorescence discrimination of coexisting lipid phase types, but thus far have focused on coexisting optically resolvable fluid phases in simple lipid mixtures. Here we demonstrate that giant plasma membrane vesicles (GPMVs) or blebs formed from the plasma membranes of cultured mammalian cells can also segregate into micrometer-scale fluid phase domains. Phase segregation temperatures are widely spread, with the vast majority of GPMVs found to form optically resolvable domains only at temperatures below ≈25°C. At 37°C, these GPMV membranes are almost exclusively optically homogenous. At room temperature, we find diagnostic lipid phase fluorophore partitioning preferences in GPMVs analogous to the partitioning behavior now established in model membrane systems with liquid-ordered and liquid-disordered fluid phase coexistence. We image these GPMVs for direct visual characterization of protein partitioning between coexisting liquid-ordered-like and liquid-disordered-like membrane phases in the absence of detergent perturbation. For example, we find that the transmembrane IgE receptor FcεRI preferentially segregates into liquid-disordered-like phases, and we report the partitioning of additional well known membrane associated proteins. Thus, GPMVs now provide an effective approach to characterize biological membrane heterogeneities.

Dynamic clustered distribution of hemagglutinin resolved at 40 nm in living cell membranes discriminates between raft theories

Organization in biological membranes spans many orders of magnitude in length scale, but limited resolution in far-field light microscopy has impeded distinction between numerous biomembrane models. One canonical example of a heterogeneously distributed membrane protein is hemagglutinin (HA) from influenza virus, which is associated with controversial cholesterol-rich lipid rafts. Using fluorescence photoactivation localization microscopy, we are able to image distributions of tens of thousands of HA molecules with subdiffraction resolution (≈40 nm) in live and fixed fibroblasts. HA molecules form irregular clusters on length scales from ≈40 nm up to many micrometers, consistent with results from electron microscopy. In live cells, the dynamics of HA molecules within clusters is observed and quantified to determine an effective diffusion coefficient. The results are interpreted in terms of several established models of biological membranes.

Focal volume optics and experimental artifacts in confocal fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) can provide a wealth of information about biological and chemical systems on a broad range of time scales (<1 micros to >1 s). Numerical modeling of the FCS observation volume combined with measurements has revealed, however, that the standard assumption of a three-dimensional Gaussian FCS observation volume is not a valid approximation under many common measurement conditions. As a result, the FCS autocorrelation will contain significant, systematic artifacts that are most severe with confocal optics when using a large detector aperture and aperture-limited illumination. These optical artifacts manifest themselves in the fluorescence correlation as an apparent additional exponential component or diffusing species with significant (>30%) amplitude that can imply extraneous kinetics, shift the measured diffusion time by as much as approximately 80%, and cause the axial ratio to diverge. Artifacts can be minimized or virtually eliminated by using a small confocal detector aperture, underfilled objective back-aperture, or two-photon excitation. However, using a detector aperture that is smaller or larger than the optimal value (approximately 4.5 optical units) greatly reduces both the count rate per molecule and the signal-to-noise ratio. Thus, there is a tradeoff between optimizing signal-to-noise and reducing experimental artifacts in one-photon FCS.

Precisely and accurately localizing single emitters in fluorescence microscopy

Methods based on single-molecule localization and photophysics have brought nanoscale imaging with visible light into reach. This has enabled single-particle tracking applications for studying the dynamics of molecules and nanoparticles and contributed to the recent revolution in super-resolution localization microscopy techniques. Crucial to the optimization of such methods are the precision and accuracy with which single fluorophores and nanoparticles can be localized. We present a lucid synthesis of the developments on this localization precision and accuracy and their practical implications in order to guide the increasing number of researchers using single-particle tracking and super-resolution localization microscopy.

Imaging biological structures with fluorescence photoactivation localization microscopy

Fluorescence photoactivation localization microscopy (FPALM) images biological structures with subdiffraction-limited resolution. With repeated cycles of activation, readout and bleaching, large numbers of photoactivatable probes can be precisely localized to obtain a map (image) of labeled molecules with an effective resolution of tens of nanometers. FPALM has been applied to a variety of biological imaging applications, including membrane, cytoskeletal and cytosolic proteins in fixed and living cells. Molecular motions can be quantified. FPALM can also be applied to nonbiological samples, which can be labeled with photoactivatable probes. With emphasis on cellular imaging, we describe here the adaptation of a conventional widefield fluorescence microscope for FPALM and present step-by-step procedures to successfully obtain and analyze FPALM images. The fundamentals of this protocol may also be applicable to users of similar imaging techniques that apply localization of photoactivatable probes to achieve super-resolution. Once alignment of the setup has been completed, data acquisitions can be obtained in approximately 1–30 min and analyzed in approximately 0.5–4 h.

Nanoscale imaging of molecular positions and anisotropies.

Knowledge of the orientation of molecules within biological structures is crucial to understanding the mechanisms of cell function. We present a method to image simultaneously the positions and fluorescence anisotropies of large numbers of single molecules with nanometer lateral resolution within a sample. Based on a simple modification of fluorescence photoactivation localization microscopy (FPALM), polarization (P)-FPALM does not compromise speed or sensitivity. We show results for mouse fibroblasts expressing Dendra2-actin or Dendra2-hemagglutinin.

Multiphoton molecular spectroscopy and excited-state dynamics of enhanced green fluorescent protein (EGFP): acid–base specificity☆

Green fluorescent protein (GFP), isolated from Aequorea victoria jellyfish, has been used extensively as a noninvasive intracellular pH indicator and site-specific fluorescent marker in biochemistry, cell biology, and molecular genetics. Numerous mutations, aimed at optimizing spectroscopic and thermodynamic properties of GFP, have been created for different applications. Fluorescence correlation spectroscopy (FCS) reveals that the enhanced green fluorescent protein mutant (EGFP; S65T/F64L) undergoes external proton exchange with the buffer on ∼45–300 μs time scale with pKa=5.8±0.1 [Proc. Natl. Acad. Sci. USA 95 (1998) 13573]. This contribution represents a comprehensive characterization of pH and excitation mode (wavelength, one and two photon (2P)) effects on the spectroscopy, excited-state dynamics, and rotational mobility of EGFP aiming at elucidating the significant electronic states of this molecular system. EGFP exhibits a large 2P action cross-section and, therefore, is well suited for intracellular imaging using 2P fluorescence microscopy.

The influence of nearest neighbors on the rate and pattern of spontaneous point mutations

SummaryThe numbers and local sequence environments of the two types of substitution mutation plus additions and deletions have been obtained directly in this study from differences between a large number of extant primate gene and pseudogene sequences. A total of 3786 mutations were scored in regions where similarities between pseudogene and corresponding gene sequences is ≥ 85%, comprising ∼30% of the pseudogene database of 80,584 bp. The pattern of mutations obtained in this fashion is almost identical to that obtained by Li et al. (1984) using a slightly different, more direct approach and with a smaller database. When mutations were scored, the neighbor pairs on the 5′ and 3′ sides were also noted, leading to a large 16 × 12 matrix of transitions and transversions. Biases of varying magnitude are found in the rates of substitution of the same base pair in different local sequence environments. The overall order for the effect of the 5′ neighbor on the rates of substitution mutation of a pyrimidine is A > C ≫ T > G, and G > A > T > C for the 3′ neighbor; where these results represent the average of substitution rates for the complement purine with complement neighbors of bases ordered above. The order for the 3′ neighbor is essentially the same for the two transitions and most of the four transversions as well; however, the order for the 5′ neighbor is more variable. The overall rate for the C · G → T · A transition is not unusual, however the presence of a 3′ neighboring G · C pair boosts the rate substantially, presumably due to specific cytosine methylation of the CG doublet in primate DNAs. The rate of the T · A → C · G transition is also well above average when the 3′ neighbor is an A · T, and to a lesser extent a G · C, pair. The latter bias is typical in that it reflects the association of alternating pyrimidine-purine sequences with increasing mutation rates. The substitution of the pyrimidine in a 5′ purine-pyrimi-dine-purine3′ sequence generally occurs much faster than in a pyrimidine tract and points to the local conformation as a major determining factor of the substitution rate. An apparent inverse relationship is found between starting and product doublet frequencies of base pairs undergoing mutations with specific 3′ neighbors, indicating that differences in intrinsic substitution rates of base pairs with specific neighbors are a key factor in producing the familiar biases of nearest-neighbor frequencies.