Dynamic breast MRI with spiral trajectories: 3D versus 2D.

A three-dimensional (3)D spiral sequence was developed for dynamic breast magnetic resonance (MR) imaging with much improved image quality. Partial Z phase encoding was applied to obtain thinner slices for a coverage of the whole breast. Comparison between the 3D and a previously developed multi-slice 2D spiral sequences was performed on ten healthy volunteers without contrast and five breast patients with gadolinium-diethylene triamine pentaacetic acid (Gd-DTPA). The 3D spiral images had significantly less off-resonance blurring and spiral artifacts. With a small compromise on temporal resolution (7.7 seconds in 2D and 10.6 seconds in 3D), we obtained 32 interpolated 3-5 mm slices (with 20 Z phase encodes) for a full coverage of 10-16 cm breast with the same 1 x 1 mm2 in-plane resolution as the 2D sequence, which had 12 8-13 mm slices. Contrast between glandular and soft tissue in normal breasts was increased by about 25%. The reduced repetition time in the 3D spiral acquisition led to an increased Gd-enhanced signal. The difference between the enhancement of malignant and benign lesions increased by sevenfold. We expect that this new development could lead to improved specificity in characterizing breast lesions using MR imaging.

Immunohistochemical evidence of down-regulation of mu-opioid receptor after chronic PL-017 in rats.

In a previous study, mu-opioid receptor binding was decreased by chronic treatment of rats with a mu-opioid receptor-selective agonist [CH3Phe3, D-Pro4]morphiceptin (PL-017) [Tao, P.L., Lee, H.Y., Chang, L.R., Loh, H.H., 1990. Decrease in mu-opioid receptor binding capacity in rat brain after chronic PL-017 treatment. Brain Res. 526, 270-275]. However, there was a lack of correlation between the time course of receptor down-regulation and the loss of pharmacological effects of the drug. In the current study, we used immunohistochemistry to reinvestigate this issue. Male Sprague-Dawley rats were chronically treated with PL-017 i.c.v. for 1, 3 or 5 days, using an escalating dosage paradigm (0.75-6.0 microg), which resulted in a 1.4 to 32-fold increase in the AD50. Rat brains were removed, frozen, coronally sectioned (14 microm) and processed for mu-, delta- or kappa-opioid receptor immunohistochemistry by the avidin-biotin complex (ABC) method. Significant decreases in OP3 immunodensity were found in many brain regions which are enriched with OP3 after chronic treatment of PL-017. Time-dependent decreases in OP3 were detected and reached a plateau around 3 days of PL-017 treatment. No significant change in OP1 or OP2 immunodensity after chronic treatment with PL-017 was found. Our conclusion is that chronic treatment with PL-017 of rats selectively down-regulates mu-opioid receptors in the brain. This may be an important mechanism for PL-017 tolerance.

Three-dimensional structures and contexts associated with recurrent amino acid sequence patterns.

We have used cluster analysis to identify recurring sequence patterns that transcend protein family boundaries. A subset of these patterns occur predominantly in a single type of local structure in proteins. Here we characterize the three-dimensional structures and contexts in which these sequence patterns occur, with particular attention to the interactions responsible for their structural selectivity.

Practical image restoration of thick biological specimens using multiple focus levels in transmission electron microscopy.

Three-dimensional electron tomographic studies of thick specimens such as cellular organelles or supramolecular structures require accurate interpretations of transmission electron micrograph intensities. In addition to microscope lens aberrations, thick specimen imaging is complicated by additional distortions resulting from multiple elastic and inelastic scattering. Extensive analysis of the mechanism of image formation using electron energy-loss spectroscopy and imaging as well as exit wavefront reconstruction demonstrated that multiple scattering does not contribute to the coherent component of the exit wave (Han et al., 1996, 1995). Although exit wavefront restored images showed enhanced contrast and resolution, that technique, which requires the collection of more than 30 images at different focus levels, is not practical for routine data collection in 3D electron tomography, where usually over 100 projection views are required for each reconstruction. Using a 0.7-micron-thick specimen imaged at 200 keV, the accuracy of reconstructions using small numbers of defocused images and a simple linear filter (Schiske, 1968) was assessed by comparison to the complete exit wave restoration. We demonstrate that only four optimal focus levels are required to effectively restore the coherent component (deviation 5.1%). By contrast, the optimal single image (zero defocus) shows a 25.5% deviation to the exit wave restoration. Two pairs of under- and over-defocus images should be taken: one pair at quite high defocus (> 10 microns) to differentiate the coherent (single elastic scattering) from the incoherent (multiple elastic and inelastic scattering) components, and the second pair to optimize information content at the highest desired resolution (e.g., 5 microns for (2.5 nm)-1 resolution). We also propose a new interpretation of the restored amplitude and phase components where the specimen mass-density is proportional to the logarithm of the amplitude component and linearly related to the phase component. This approach should greatly facilitate the collection of high resolution tomographic data from thick samples.

Optimal strategies for imaging thick biological specimens: exit wavefront reconstruction and energy-filtered imaging.

In transmission electron microscopy (TEM) of thick biological specimens, the relationship between the recorded image intensities and the projected specimen mass density is distorted by incoherent electron-specimen interactions and aberrations of the objective lens. It is highly desirable to develop a strategy for maximizing and extracting the coherent image component, thereby allowing the projected specimen mass density to be directly related to image intensities. For this purpose, we previously used exit wavefront reconstruction to understand the nature of image formation for thick biological specimens in conventional TEM. Because electron energy-loss filtered imaging allows the contributions of inelastically scattered electrons to be removed, it is potentially advantageous for imaging thick, biological samples. In this paper, exit wavefront reconstruction is used to quantitatively analyse the imaging properties of an energy-filtered microscope and to assess its utility for thick-section microscopy. We found that for imaging thick biological specimens (> 0.5 microns) at 200 keV, only elastically scattered electrons contribute to the coherent image component. Surprisingly little coherent transfer was seen when using energy-filtering at the most probable energy loss (in this case at the first plasmon energy-loss peak). Furthermore, the use of zero-loss filtering in combination with exit wavefront reconstruction is considerably more effective at removing the effects of multiple elastic and inelastic scattering and microscope objective lens aberrations than either technique by itself. Optimization of the zero-loss signal requires operation at intermediate to high primary voltages (> 200 keV). These results have important implications for the accurate recording of images of thick biological specimens as, for instance, in electron microscope tomography.

Local sequence-structure correlations in proteins.

Considerable progress has been made in understanding the relationship between local amino acid sequence and local protein structure. Recent highlights include numerous studies of the structures adopted by short peptides, new approaches to correlating sequence patterns with structure patterns, and folding simulations using simple potentials.

Global properties of the mapping between local amino acid sequence and local structure in proteins.

Local protein structure prediction efforts have consistently failed to exceed approximately 70% accuracy. We characterize the degeneracy of the mapping from local sequence to local structure responsible for this failure by investigating the extent to which similar sequence segments found in different proteins adopt similar three-dimensional structures. Sequence segments 3-15 residues in length from 154 different protein families are partitioned into neighborhoods containing segments with similar sequences using cluster analysis. The consistency of the sequence-to-structure mapping is assessed by comparing the local structures adopted by sequence segments in the same neighborhood in proteins of known structure. In the 154 families, 45% and 28% of the positions occur in neighborhoods in which one and two local structures predominate, respectively. The sequence patterns that characterize the neighborhoods in the first class probably include virtually all of the short sequence motifs in proteins that consistently occur in a particular local structure. These patterns, many of which occur in transitions between secondary structural elements, are an interesting combination of previously studied and novel motifs. The identification of sequence patterns that consistently occur in one or a small number of local structures in proteins should contribute to the prediction of protein structure from sequence.

Recurring local sequence motifs in proteins.

We describe a completely automated approach to identifying local sequence motifs that transcend protein family boundaries. Cluster analysis is used to identify recurring patterns of variation at single positions and in short segments of contiguous positions in multiple sequence alignments for a non-redundant set of protein families. Parallel experiments on simulated data sets constructed with the overall residue frequencies of proteins but not the inter-residue correlations show that naturally occurring protein sequences are significantly more clustered than the corresponding random sequences for window lengths ranging from one to 13 contiguous positions. The patterns of variation at single positions are not in general surprising: chemically similar amino acids tend to be grouped together. More interesting patterns emerge as the window length increases. The patterns of variation for longer window lengths are in part recognizable patterns of hydrophobic and hydrophilic residues, and in part less obvious combinations. A particularly interesting class of patterns features highly conserved glycine residues. The patterns provide a means to abstract the information contained in multiple sequence alignments and may be useful for comparison of distantly related sequences or sequence families and for protein structure prediction.

Mechanism of image formation for thick biological specimens: exit wavefront reconstruction and electron energy-loss spectroscopic imaging.

With increasing frequency, cellular organelles and nuclear structures are being investigated at high resolution using electron microscopic tomography of thick sections (0.3-1.0 microns). In order to reconstruct the structures in three dimensions accurately from the observed image intensities, it is essential to understand the relationship between the image intensity and the specimen mass density. The imaging of thick specimens is complicated by the large fraction of multiple scattering which gives rise to incoherent and partially coherent image components. Here we investigate the mechanism of image formation for thick biological specimens at 200 and 300 keV in order to resolve the coherent scattering component from the incoherent (multiple scattering) components. Two techniques were used: electron energy-loss spectroscopic imaging (ESI) and exit wavefront reconstruction using a through-focus series. Although it is commonly assumed that image formation of thick specimens is dominated by amplitude (absorption) contrast, we have found that for conventionally stained biological specimens phase contrast contributes significantly, and that at resolutions better than approximately 10 nm, superposed phase contrast dominates. It is shown that the decrease in coherent scattering with specimen thickness is directly related to the increase in multiple scattering. It is further shown that exit wavefront reconstruction can exclude the microscope aberrations as well as the multiple scattering component from the image formation. Since most of the inelastic scattering with these thick specimens is actually multiple inelastic scattering, it is demonstrated that exit wavefront reconstruction can act as a partial energy filter. By virtue of excluding the multiple scattering, the 'restored' images display enhanced contrast and resolution. These findings have direct implications for the three-dimensional reconstruction of thick biological specimens, where a simple direct relationship between image intensity and mass density was assumed, and the aberrations were left uncorrected.