Observations on the behavior of vitreous ice at approximately 82 and approximately 12 K

In an attempt to determine why cooling with liquid helium actually proved disadvantageous in our electron cryotomography experiments, further tests were performed to explore the differences in vitreous ice at approximately 82 and approximately 12 K. Electron diffraction patterns showed clearly that the vitreous ice of interest in biological electron cryomicroscopy (i.e., plunge-frozen, buffered protein solutions) does indeed collapse into a higher density phase when irradiated with as few as 2-3 e-/A2 at approximately 12 K. The high density phase spontaneously expanded back to a state resembling the original, low density phase over a period of hours at approximately 82 K. Movements of gold fiducials and changes in the lengths of tunnels drilled through the ice confirmed these phase changes, and also revealed gross changes in the concavity of the ice layer spanning circular holes in the carbon support. Brief warmup-cooldown cycles from approximately 12 to approximately 82 K and back, as would be required by the flip-flop cryorotation stage, did not induce a global phase change, but did allow certain local strains to relax. Several observations including the rates of tunnel collapse and the production of beam footprints suggested that the high density phase flows more readily in response to irradiation. Finally, the patterns of bubbling were different at the two temperatures. It is concluded that the collapse of vitreous ice at approximately 12 K around macromolecules is too rapid to account alone for the problematic loss of contrast seen, which must instead be due to secondary effects such as changes in the mobility of radiolytic fragments and water.     

Radiation damage effects at four specimen temperatures from 4 to 100 K

Radiation damage is the primary factor that limits resolution in electron cryo-microscopy (cryo-EM) of frozen-hydrated biological samples. Negative effects of radiation damage are attenuated by cooling specimens to cryogenic temperatures using liquid nitrogen or liquid helium. We have examined the relationship between specimen temperature and radiation damage across a broad spectrum of resolution by analyzing images of frozen-hydrated catalase crystal at four specimen temperatures: 4, 25, 42, and 100 K. For each temperature, “exposure series” were collected consisting of consecutive images of the same area of sample, each with 10 e^?/Ų exposure per image. Radiation damage effects were evaluated by examining the correlation between cumulative exposure and normalized amplitudes or IQ values of Bragg peaks across a broad range of resolution (4.0–173.5 Å). Results indicate that for sub-nanometer resolution, liquid nitrogen specimen temperature (100 K) provides the most consistent high-quality data while yielding statistically equivalent protection from radiation damage compared to the three lower temperatures. At lower resolution, suitable for tomography, intermediate temperatures (25 or 42 K) may provide a modest improvement in cryo-protection without introducing deleterious effects evident at 4 K.     

From proteomic inventory to architecture

Electron tomography can provide three-dimensional reconstructions of large pleomorphic structures at molecular resolution. While the principles of electron tomography have been known for decades, its use has gathered momentum only in recent years. Technological advances have made it possible to apply it to ice-embedded biological material (cryotomography), thereby ensuring a close-to-life preservation of the samples. In combination with advanced computational methods, such as molecular identification based on pattern recognition, it is a promising approach to comprehensively map macromolecular architecture inside organelles and cells and to visualize macromolecules at work in their natural environment.     

Conical tomography of freeze-fracture replicas: a method for the study of integral membrane proteins inserted in phospholipid bilayers

We have used conical tomography to study the structure of integral proteins in their phospholipid bilayer environments. Complete conical series were collected from replicas of the water channel aquaporin-0 (AQP0), a 6.6 nm side tetramer with a molecular weight of approximately 120 kDa that was purified and reconstituted in liposomes. The replicas were tilted at 38 degrees , 50 degrees or 55 degrees and rotated by 2.5 degrees , 4 degrees , or 5 degrees increments until completing 360 degrees turns. The elliptical paths of between 6 and 12 freeze-fracture particles aligned the images to a common coordinate system. Using the weighted back projection algorithm, small volumes of the replicas were independently reconstructed to reconstitute the field. Using the Fourier Shell Correlation computed from reconstructions of even and odd projections of the series, we estimated a resolution of 2-3 nm, a value that was close to the thickness of the replica (approximately 1.5 nm). The 3D reconstructions exhibited isotropic resolution along the x-y plane, which simplified the analysis of particles oriented randomly in the membrane plane. In contrast to reconstructions from single particles imaged using random conical tilt [J. Mol. Biol. 325 (2003) 210], the reconstructions using conical tomography allowed the size and shape of individual particles representing the AQP0 channel to be identified without averaging or imposing symmetry. In conclusion, the reconstruction of freeze-fracture replicas with electron tomography has provided a novel experimental approach for the study of integral proteins inserted in phospholipid bilayers.     

Electron cryotomography sample preparation using the Vitrobot

Electron cryotomography is the highest-resolution structural technique currently available that can be applied to unique objects such as flexible large protein complexes, irregular viruses, organelles and small cells. Specimens are preserved in a near-native, 'frozen-hydrated' state by vitrification. The thickness of the vitreous ice must be optimized for each specimen, and gold fiducials are typically added to facilitate image alignment. Here, we describe in detail our protocols for electron cryotomography sample preparation including (i) introduction of fiducial markers into the sample and (ii) sample vitrification. Because we almost exclusively use an automated, climate-controlled plunge-freezing device (the FEI Vitrobot) to vitrify our samples, we discuss its operation and parameters in detail. A session in which eight grids are prepared takes 1.5-2 h.     

Effects of radiation damage with 400-kV electrons on frozen, hydrated actin bundles.

Electron images can be used to provide amplitudes and phases for the structural determination of biological specimens. Radiation damage limits the amount of structural information retrievable by computer processing. A 400-kV electron microscope was used to investigate radiation damage effects on frozen, hydrated actin bundles kept at -168 degrees C. The quality of phases within and among images in a damage series was evaluated quantitatively out to 16 A resolution. It was found that the phases of structure factors with good signal-to-noise ratio (IQ less than or equal to 4) can be reliably retrieved from images taken at a cumulative dose of at least 25 electrons/A2.     

Determination of myo-inositol in biological samples by liquid chromatography-mass spectrometry

A high-performance liquid chromatographic method using liquid-liquid extraction was developed for the determination of 1-(3-fluoro-4-hydroxy-5-mercaptomethyl-tetrahydrofuran-2-yl)-5-methyl-1H-pyrimidine-2,4-dione (l-FMAUS; I) in rat plasma and urine. A 100 microl aliquot of distilled water containing l-cysteine (100 mg/ml) was added to a 100 microl aliquot of biological sample. l-Cysteine was employed to protect binding between the 5'-thiol of I and protein in the biological sample. After vortex-mixing for 30s and adding a 50 microl aliquot of the mobile phase containing the internal standard (10 microg/ml of 3-aminophenyl sulfone), 1 ml of ethyl acetate was used for extraction. After vortex-mixing, centrifugation, and evaporating the ethyl acetate, the residue was reconstituted with a 100 microl aliquot of the mobile phase. A 50 microl aliquot was injected onto a C(18) reversed-phase column. The mobile phases, 50 mM KH(2)PO(4) (pH = 2.5):acetonitrile (85:15, v/v) for rat plasma and 50 mM KH(2)PO(4) (pH 2.5):acetonitrile:methanol (85:10:5, v/v/v) for urine samples, were run at a flow-rate of 1.2 ml/min. The column effluent was monitored by an ultraviolet detector set at 265 nm. The retention times for I and the internal standard were approximately 9.7 and 12.5 min, respectively, in plasma samples and the corresponding values in urine samples were 16.8 and 14.9 min. The quantitation limits of I in rat plasma and urine were 0.1 and 0.5 microg/ml, respectively.     

Applications of a bilateral denoising filter in biological electron microscopy

Due to the sensitivity of biological sample to the radiation damage, the low dose imaging conditions used for electron microscopy result in extremely noisy images. The processes of digitization, image alignment, and 3D reconstruction also introduce additional sources of noise in the final 3D structure. In this paper, we investigate the effectiveness of a bilateral denoising filter in various biological electron microscopy applications. In contrast to the conventional low pass filters, which inevitably smooth out both noise and structural features simultaneously, we found that bilateral filter holds a distinct advantage in being capable of effectively suppressing noise without blurring the high resolution details. In as much, we have applied this technique to individual micrographs, entire 3D reconstructions, segmented proteins, and tomographic reconstructions.     

Characterizing the secondary hydration shell on hydrated myoglobin, hemoglobin, and lysozyme powders by its vitrification behavior on cooling and its calorimetric glass-->liquid transition and crystallization behavior on reheating.

For hydrated metmyoglobin, methemoglobin, and lysozyme powders, the freezable water fraction of between approximately 0.3-0.4 g water/g protein up to approximately 0.7-0.8 g water/g protein has been fully vitrified by cooling at rates up to approximately 1500 K min-1 and the influence of cooling rate characterized by x-ray diffractograms. This vitreous but freezable water fraction started to crystallize at approximately 210 K to cubic ice and at approximately 240 K to hexagonal ice. Measurements by differential scanning calorimetry have shown that this vitreous but freezable water fraction undergoes, on reheating at a rate of 30 K min-1, a glass-->liquid transition with an onset temperature of between approximately 164 and approximately 174 K, with a width of between approximately 9 and approximately 16 degrees and an increase in heat capacity of between approximately 20 and approximately 40 J K-1 (mol of freezable water)-1 but that the glass transition disappears upon crystallization of the freezable water. These calorimetric features are similar to those of water imbibed in the pores of a synthetic hydrogel but very different from those of glassy bulk water. The difference to glassy bulk water's properties is attributed to hydrophilic interaction and H-bonding of the macromolecules' segments with the freezable water fraction, which thereby becomes dynamically modified. Abrupt increase in minimal or critical cooling rate necessary for complete vitrification is observed at approximately 0.7-0.8 g water/g protein, which is attributed to an abrupt increase of water's mobility, and it is remarkably close to the threshold value of water's mobility on a hydrated protein reported by Kimmich et al. (1990, Biophys. J. 58:1183). The hydration level of approximately 0.7-0.8 g water/g protein is approximately that necessary for completing the secondary hydration shell.     

Local refinement: an attempt to correct for shrinkage and distortion in electron tomography

A critical problem in electron tomography is the deformation of the specimen due to radiation, or "shrinkage," which interferes with image alignment and thereby limits resolution. Here, we describe a general strategy for refining preliminary reconstructions which allows the damage due to the shrinkage of plastic-embedded thin sectioned specimens (50-80 nm) to be corrected. The basic steps of the strategy involve: (a) the partition of the preliminary reconstruction into sub-volumes; (b) the extraction of corresponding sub-areas for each sub-volume from the micrographs of the tilt series; (c) the re-projection of each sub-volume according to the orientation parameters; and (d) the refinement of these parameters by correlating each sub-area to the corresponding computed projection. We tested the strategy by refining chemical synapses reconstructed from series imaged with conical, double and single tilt geometries. The results gathered with local refinement were evaluated by visually inspecting the structure of biological membranes in the maps. In an effort to quantify these improvements, we studied the refined maps using correlation criteria and mapped the corrections applied to the orientation parameters in each sub-volume of the reconstruction. Simulation experiments complemented the data gathered by correlation analysis. Based on these criteria, we concluded that local refinement significantly improves the overall quality of the reconstructions of chemical synapses calculated from series imaged with conical and double tilt geometries.     

Use of frozen-hydrated axonemes to assess imaging parameters and resolution limits in cryoelectron tomography

Using a 400-kV cryoelectron microscope, we have obtained tomographic reconstructions of frozen-hydrated sea urchin axonemes with 8-10-nm resolution, as assessed by detection of characteristic components including doublet microtubules, radial spokes, central sheath projections, and outer dynein arms. We did not detect the inner dynein arms or the microtubule lattice. The 1/(8 nm) and 1/(16 nm) layer lines are consistently present in power spectra of both projection images and tomographic reconstructions. Strength and detection of the layer lines are dependent upon total electron dose and defocus. Both layer lines are surprisingly resistant to electron doses of up to 11000 electrons/nm(2). We present a summary of resolution considerations in cryoelectron tomography and conclude that the fundamental limitation is the total electron dose required for statistical significance. The electron dose can be fractionated among the numerous angular views in a tomographic data set, but there is an unavoidable fourth-power dependence of total dose on target resolution. Since higher-resolution features are more beam-sensitive, this dose requirement places an ultimate limit on the resolution of individual tomographic reconstructions. Instrumental and computational strategies to circumvent this limitation are discussed.     

Theoretical analysis of specimen cooling rate during impact freezing and liquid-jet freezing of freeze-etch specimens.

We have carried out a theoretical analysis of specimen cooling rate under ideal conditions during impact freezing and liquid-jet freezing. The analysis shows that use of liquid helium instead of liquid nitrogen as cooling medium during impact freezing results in an increase in a specimen cooling rate of no more than 30-40%. We have further shown that when both impact freezing and liquid-jet freezing are conducted at liquid nitrogen temperature, the two methods give approximately the same specimen cooling rate under ideal conditions except for a thin outer layer of the specimen. In this region impact freezing yields the highest cooling rate.     

Refractive index measurement using single fiber reflectance spectroscopy

A method using single fiber reflectance spectroscopy to measure the refractive indices of transparent and turbid media over a broad wavelength range is presented and tested. For transparent liquid samples, the accuracy is within 0.2%, and the accuracy increases with increasing wavelength. For liquid turbid media, the accuracy is within 0.3% and increases with decreasing wavelength. For solid turbid samples, such as human skin, the accuracy critically depends on the optical contact between the fiber and sample surface. It is demonstrated that this technique has the potential to measure refractive indices of biological tissue in vivo.      

Photobiological and thermal effects of photoactivating UVA light doses on cell cultures.

While near-ultraviolet light has been widely used to photoactivate fluorophores and caged compounds in cells, little is known of the long-term biological effects of this light. UVA (315-400 nm) photoactivating light has been well characterized in short-term cell studies and is now being employed in higher doses to control longer-duration phenomena (e.g. gene expression). Annexin V-Cy5/propidium iodide apoptosis flow cytometry assays were used to determine responses of HeLa cells to doses of UVA light up to 23.85 J cm(-2). Cells seeded at low densities had higher percentages of apoptosis and necrosis and were also more susceptible to UVA damage than cells seeded at higher densities. The dose to induce apoptosis and death in 50% of the cells (dose(1/2)) was determined for two different commercially available UVA light sources: 7.6 J cm(-2) for the GreenSpot photocuring system and 2.52 J cm(-2) for the BlakRay lamp. All BlakRay doses tested had significant cellular responses, whereas no significant cellular responses were found for doses below 1.6 J cm(-2) from the GreenSpot light source. A temperature control and measurement system was used to determine direct heating from the UVA sources and also the effect that cooling cell cultures during photoexposure has on minimizing cell damage. Cooling during the BlakRay photoexposure significantly reduced the percentage of necrotic cells, but there was no significant difference for cooling during photoactivation with the GreenSpot. Differences in cell responses to similar UVA doses of different intensities suggest that photoduration should be considered along with total dose and thermal conditions in photoactivation studies.     

Determination of an arylacetamide antiarrhythmic in rat blood and tissues using reversed-phase high-performance liquid chromatography

A method was developed for quantification of (+)-trans-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzo[b- ]thiophene-4-acetamide (compound I), an antiarrhythmic drug, in rat whole blood, heart, brain, liver and skeletal muscle. Blood and tissue samples were homogenized and purified by chemical extraction. Chromatographic separations were achieved using reversed-phase high-performance liquid chromatography (HPLC) coupled with UV detection (215 nm). Drug recoveries from the extraction procedure ranged from 77 to 90%. Within- and between-day reproducibility of peak area (coefficient of variation) ranged from 1.1 to 15.7%. The detection limit was 80–200 ng/ml (in a 500-μl extracted solution) depending on the type of biological sample. This method was used in a pharmacokinetic study of compound I disposition in rats after a bolus intravenous dose of 3.1 mg/kg.     

Radiation damage in tripalmitin layers studied by means of infrared spectroscopy and electron microscopy.

Structural deteriorations in biomembranes, as inevitably induced while structural information is gathered by electron optical methods, were evaluated by infrared spectroscopy. Tripalmitin model membranes were irradiated with 100 keV-electrons in an electron microscope. The intensity decay of group vibrations over the dose reveals the sequence of damage in the polar and nonpolar part of the molecule. The C-C backbone, being the most important structural feature, shows a significant latency effect up to 0.6 e-/A2 and is completely disordered by 3 e-/A2, corresponding to about three inelastic processes per molecule.     

Analyzing indirect secondary electron contrast of unstained bacteriophage T4 based on SEM images and Monte Carlo simulations

The indirect secondary electron contrast (ISEC) condition of the scanning electron microscopy (SEM) produces high contrast detection with minimal damage of unstained biological samples mounted under a thin carbon film. The high contrast image is created by a secondary electron signal produced under the carbon film by a low acceleration voltage. Here, we show that ISEC condition is clearly able to detect unstained bacteriophage T4 under a thin carbon film (10-15 nm) by using high-resolution field emission (FE) SEM. The results show that FE-SEM provides higher resolution than thermionic emission SEM. Furthermore, we investigated the scattered electron area within the carbon film under ISEC conditions using Monte Carlo simulation. The simulations indicated that the image resolution difference is related to the scattering width in the carbon film and the electron beam spot size. Using ISEC conditions on unstained virus samples would produce low electronic damage, because the electron beam does not directly irradiate the sample. In addition to the routine analysis, this method can be utilized for structural analysis of various biological samples like viruses, bacteria, and protein complexes.     

Contribution of Starling and lymphatic flows to pleural liquid exchanges in anesthetized rabbits

We studied the time course of volume and protein reabsorption of a 2-ml hydrothorax using whole (WP) or diluted (DP) homologous plasma injected into the right pleural cavity in anesthetized spontaneously breathing supine rabbits. Animals were killed at 5 (WP, n = 4; DP, n = 3), 36 (WP, n = 3; DP, n = 4), 55 (WP, n = 4), 90 (WP, n = 8; DP, n = 4), and 150 (WP, n = 4; DP, n = 5) min after the injection. The volume and protein content of the pleural liquid in control conditions (n = 12) amounted to 0.35 +/- 0.015 (SE) ml/kg and 1.8 +/- 0.27 g/100 ml, respectively, which are not significantly different at 90 min (n = 7). Pleural liquid volume decreased at a similar rate during WP or DP reabsorption according to the equation V = 0.84 +/- 0.05 X e-0.02t, with net reabsorptive flow expressed as dV/dt. The globulin quantity (Q) of the pleural liquid for WP and DP, respectively, decreased according to the equations Qwp = 1 + 1.5 X e-0.04t and Qdp = 0.7 + 0.6 X e-0.03t. Assuming a major lymphatic globulin clearance and no filtration into the cavity, we obtained lymph flow using the equation VL = dQ/dt X l/C where dQ/dt is calculated from the equations for Qwp and Qdp and C represents globulin concentration. The Starling flow (Vs) was then calculated by the equation Vs = dV/dt-VL. With increasing time, lymph flow was found to decrease progressively and was not significantly different from net flow with DP, which implied a Starling flow value of zero. During WP reabsorption, lymph flow initially exceeded the net flow, with the difference disappearing at approximately 60 min; accordingly, Starling filtration flow decreased progressively, becoming zero at the same time.     

Determination of EETs using microbore liquid chromatography with fluorescence detection

Epoxyeicosatrienoic acids (EETs) are cytochrome P-450 metabolites of arachidonic acid involved in the regulation of vascular tone. The method of microbore column high-performance liquid chromatography with fluorescence detection was developed to determine 14,15-EET, 11, 12-EET, and the mixture of 8,9-EET and 5,6-EET. Tridecanoic acid (TA) was used as an internal standard. EETs were reacted with 2-(2, 3-naphthalimino)ethyl trifluoromethanesulfonate (NT) to form highly fluorescent derivatives. A C(18) microbore column and a water-acetonitrile mobile phase were used for separation. Samples were excited at 259 nm, and the fluorescence was detected at 395 nm. The overall recoveries were 88% for EETs and 40% for TA. EETs were detected in concentrations as low as 2 pg (signal-to-noise ratio = 3). The method was used to determine the EET production from endothelial cells (ECs). Bradykinin and methacholine (10(-6) M) stimulated an increase in the production of EETs by ECs two- and fivefold, respectively. This sensitive method may be used for determination of EETs at low concentrations normally detected in complex biological samples.     

Structure determination of macromolecular assemblies by single-particle analysis of cryo-electron micrographs

A new generation of electron microscopes equipped with field emission gun electron sources and the ability to image molecules in their native environment at liquid nitrogen or helium temperatures has enabled the analysis of macromolecular structures at medium resolution (approximately 10 angstroms) and in different conformational states. The amalgamation of electron microscopy and X-ray crystallographic approaches makes it possible to solve structures in the 100-1000 angstroms size range, advancing our understanding of the function of complex assemblies. Many new structures have been solved during the past two years, including one of the smallest complexes to be determined by single-particle cryo-electron microscopy, the transferrin receptor-transferrin complex. Other notable results include the near atomic level resolution structure of the nicotinic acetylcholine receptor in helical arrays and an icosahedral virus structure with an asymmetric polymerase resolved.