Atomic force microscopy on human trabecular bone from an old woman with osteoporotic fractures

AFM images were taken of the exterior surface of a single trabecula, extracted from a human femoral head removed during surgery for a hip fracture in an old women with former fractures. The images showed a dense structure of bundled collagen fibrils banded with 67 nm periodicity. Bundles were seen to run in parallel in layers confirming the collagen structure seen by other techniques. Single collagen fibrils were seen to cross the bundles, thus forming cross-links between neighboring bundles of collagen fibrils. Some of these crossing fibrils did not have the 67 nm band pattern and their dimensions were about half compared to the neighboring collagen fibrils. Very little mineral was found on the surface of the trabecula. An AFM image of a fracture plane was also displayed. The trabecula was extracted from a region close to the hip fracture. However, there were in this case no obvious features in the images that could be linked directly to osteoporosis, but altered collagen banding and collagen protrusions may alter mechanical competence. A path to extensive studies of the nanometer scale structure of bone was demonstrated.     

DTI of trabecular bone marrow

The development of NMR diffusion imaging and diffusion tensor imaging (DTI) has offered the possibility of studying the porous structures beyond anatomical imaging. In fact, random molecular motions, within tissue components, probe tissue microstructures. Up to now, the DTI method was mainly used to investigate cerebral morphology and study white matter diseases. In this study, it has been applied to trabecular bone marrow analysis to obtain structural information on spongy bone tissue. Our first results show that DTI could represent an important tool in studying the microstructural architecture of the trabecular bone as well as the microarchitecture of porous media.     

R'2 measured in trabecular bone in vitro: relationship to trabecular separation.

Measurement of key parameters of the microstructure of trabecular bone is critical to the study of osteoporosis and bone strength. Density based methods cannot provide this information, and give only the total amount of bone present, and not its arrangement. Magnetic resonance imaging has shown the potential to provide information related to the microarchitecture of the trabecular bone matrix. Twelve samples (8 x 8 x 8 mm3 bone cubes) were cut from sheep vertebrae such that the trabeculae ran either parallel or perpendicular to each face. Detailed measurements of the structure of these bone cubes were made by histomorphometry, and compared to R'2 and R*2 measured with a spin and gradient-echo sequence, Partially Refocused Interleaved Multiple Echo, at 1.5 Tesla. The precision of the R'2 measurement (% coefficient of variation) was 8.7+/-5.1, and 7.7+/-4.3 for R*2. Uncorrected values of R'2 and R*2 were significantly correlated to density measured by quantitative computed tomography (r = 0.87, p = 0.0005, and r = 0.90, p = 0.0002, respectively), and trabecular bone area measured by histomorphometry (r = 0.80, p = 0.002, and r = 0.83, p = 0.0008, respectively). Density correction was effected by imaging the same slice of bone in two orientations (90 degrees and 0 degrees ) to the main magnetic field. For both R'2 and R*2 there was a significant difference between measurements in the 90 degrees and 0 degrees orientations (p < 0.01). The difference between the two values was used, and termed R'2net or R*2net. The net parameters were independent of bone mass. R'2net and R*2net were significantly correlated to trabecular separation (p < 0.05) with r = -0.58 and r = -0.62, respectively. These results demonstrate the ability of magnetic resonance imaging to characterize a key measure of the trabecular microstucture. An increase in trabecular separation has important biomechanical consequences in osteoporosis. This result also strengthens the hypothesis that the sensitivity of R'2 to osteoporosis-related bone changes is due to magnetic susceptibility effects in which rapid transitions between bone and marrow create local magnetic field inhomogeneities that result in an increase in R'2 values.     

Time-independent point-spread function for NMR microscopy.

The resolution of NMR microscopy is analyzed in terms of the point-spread function, PSF(r), and the equivalent k space modulation transfer function, MTF(k). The analysis is developed for NMR spin warp and projection reconstruction imaging experiments; however, the framework provided is quite general. Incoherent spin motion is analyzed to predict what limits, if any, on spatial resolution are imposed by diffusion. Previous estimates of diffusion limits at 1-5 microns were developed for specific imaging techniques, typically using a mean displacement argument. Although qualitatively correct, the quantitative predictions represent practical rather than fundamental limits. It is shown that diffusion-dependent "blurring" can be made arbitrarily small and that the practical limits are less stringent than previously thought. A major point illustrated by the PSF-MTF formulation is that the irreversible loss of coherence by randomly diffusing spins occurs faster than the physical displacement, thereby reducing their effect considerably on the frequency or phase of the net detected signal. The irreversible loss of signal due to diffusive motion will contribute to and possibly dominate the signal-to-noise limit of resolution. The resolution as measured by the width of the PSF and MTF for diffusion is shown to be independent of the signal acquisition time, and their functional forms allow selection of microscopic imaging parameters. An example of a three-dimensional spin-warp image of a green algae cell is shown with resolution of approximately 16 microns x 13 microns x 10 microns.     

Ultrahigh-spatial-resolution field-emission projection microscopy of insulating samples

The method of ultrahigh-spatial-resolution field-emission projection imaging of nonconducting tips is implemented experimentally. An image of a glass microcapillary tip was obtained for the first time by the nonscanning method with spatial resolution no worse than 20 nm.     

NMR microscopy of polyurethane foams

NMR microimaging has been applied to the characterization of foam materials, in order to understand their properties and gain deeper insight into their structures. The structure of "open cell" foams can be visualized with sufficient resolution to obtain the size and shape of cells, as well as their spatial distribution within specimens. Statistics of cell sizes have been calculated from NMR microscopy and are in good agreement with results obtained from electron microscopy. We demonstrate that the local density of foams, which are parameters largely inaccessible by other analytical methods, readily can be determined by NMR microscopy.     

Fringe projection based quantitative 3D microscopy

A fringe projection based quantitative three-dimensional microscopy (FP-3DM) is presented. The image formation for FP-3DM is formulated based on a concept of active micro stereovision. The problem of point correspondences in active stereo imaging can be solved with help of the phase measuring technique. A prototype of the FP-3DM is also established and calibration strategy for proposed FP-3DM is suggested. Some preliminary experiment results are also presented to verify this approach. The FP-3DM can provide quantitative 3D micro imaging especially for quantitative characterization of 3D microstructures.     

Ultrahigh-spatial-resolution photoelectron projection microscopy using femtosecond lasers

Ultrahigh spatial resolution of two-photon photoelectron images (as high as 3 nm, which is the best value that has been achieved to date in photoelectron microscopy with spatial resolution) is obtained when silicon nanotips are irradiated by the second harmonic of a pulsed femtosecond Ti: sapphire laser. In addition, the absolute value of the two-photon external photoeffect coefficient is measured. Zh. éksp. Teor. Fiz. 115, 1680–1688 (May 1999)     

Femtosecond laser photoelectron projection microscopy of organic nanocomplexes

Organic nanocomplexes obtained by applying an organic dye (Coumarin 153) solution onto the surface of a metal nanotip were studied by femtosecond laser photoelectron projection microscopy. The tip was a 100-nm-diameter capillary covered with a thin nickel layer. The image of a through hole of the capillary was a convenient reference for interpretation of the results. The spatial resolution achieved in these experiments was equal to about 5 nm.     

Interactive reduced FOV imaging for projection reconstruction and spiral acquisition.

MR fluoroscopy is likely to gain increasing importance for the visualization of dynamic processes such as cardiac function and for the guidance of interventional procedures. In many applications the dynamic processes are restricted to a part of the object under study making reduced field of view (rFOV) imaging desirable. The restriction to a smaller FOV can either be used to increase the spatial or the temporal resolution. In projection reconstruction (PR) and spiral imaging severe backfolding artifacts occur if a rFOV is used. In this paper efficient suppression schemes are proposed for PR- and spiral imaging to avoid backfolding artifacts. Evaluation of the proposed schemes was done on an interactive real-time MR-scanner. Cardiac function studies clearly showed the potential of this technique for PR- and spiral imaging.     

One micrometer resolution NMR microscopy

We obtained a magnetic resonance image of 1 microm resolution and 75 microm(3) voxel volume for a phantom filled with hydrocarbon oil within an hour at 14.1 T. For this work, a specially designed probe with a high sensitivity RF coil and gradient coils generating over 1000 G/cm was built. The optimal pulse sequence was analyzed in consideration of the bandwidth, diffusion coefficients, and T(1) and T(2) relaxations of the medium. The system was applied to the in vivo imaging of a geranium leaf stem to get the images of 2 microm resolution and 200 microm(3) voxel volume.     

Proton magnetic resonance microimaging of human trabecular bone

Proton magnetic resonance (1H magnetic resonance imaging (MRI)) images of human trabecular bone were acquired and discussed for two samples with different porosity. Three-dimensional 3D Spin Echo (3D SE) and Multi-Slice Multi-Echo (MSME) pulse sequences were examined. A very high slice resolution of (38 microm)2 was achieved (MSME). The intensity histograms were found useful for the characterization of the bone porosity. A spatial distribution of the spin-spin relaxation time T2 was monitored with the MSME pulse program. The work demonstrates the great potential of the proton MRI technique in the study of the trabecular bone morphology.     

Pattern projection for subpixel resolved imaging in microscopy

In this paper, we present a new approach providing super resolved images exceeding the geometrical limitation given by the detector pixel size of the imaging camera. The concept involves the projection of periodic patterns on top of the sample, which are then investigated under a microscope. Combining spatial scanning together with proper digital post-processing algorithm yields the improved geometrical resolution enhancement. This new method is especially interesting for microscopic imaging when the resolution of the detector is lower than the resolution due to diffraction.     

Laser photoelectron projection microscopy of an organic conducting polymer

A conducting organic polymer is visualized on a laser photoelectron projection microscope, which is based on Letokhov’s concept and has a nanometer spatial resolution. Photoelectron images of polyaniline (which is the most promising representative of conducting polymers) with a magnification of ∼10⁵ have been obtained when a 100-nm quartz capillary coated with a film of this material was irradiated by femtosecond laser pulses. The projection photoelectron method using 400- and 800-nm laser radiation has made it possible to directly reveal the existence of the redox heterogeneity of the organic polymer, which is due to the contact of the sections of polyaniline with different oxidation degrees and strongly affects the electric conductivity of the sample.     

Quantitative NMR microscopy on intact plants

Quantitative high resolution images on intact young maize plants were acquired by using magnetization-prepared NMR microscopy. Although the spatial resolution is low compared with that of light microscopy, the calculated spin density and T₁ maps exhibit contrasts that are in excellent agreement with photomicrographic images. The T₂ map gives image contrasts that are not visible in a usual light microscopic image. The diffusion images show an anisotropic behavior of the water self-diffusion coefficient in the vascular bundles, which can be understood by the cell morphology in this plant section. This work demonstrates that quantitative imaging on intact plant systems is possible and that long total acquisition times are no obstacle. Furthermore, the different single parameter maps give a better insight into the morphology of plants under in vivo conditions.     

Kramers-Kronig analysis of attenuation and dispersion in trabecular bone

A restricted-bandwidth form of the Kramers-Kronig dispersion relations is applied to in vitro measurements of ultrasonic attenuation and dispersion properties of trabecular bone specimens from bovine tibia. The Kramers-Kronig analysis utilizes only experimentally measured properties and avoids extrapolation of ultrasonic properties beyond the known bandwidth. Compensation for the portions of the Kramers-Kronig integrals over the unknown bandwidth is partially achieved by the method of subtractions, where a subtraction frequency acts as an adjustable parameter. Good agreement is found between experimentally measured and Kramers-Kronig reconstructed dispersions. The restricted-bandwidth approach improves upon other forms of the Kramers-Kronig relations and may provide further insight into how ultrasound interacts with trabecular bone.