Corneal confocal microscopy : a novel noninvasive test to diagnose and stratify the severity of human diabetic neuropathy

OBJECTIVE: The accurate quantification of human diabetic neuropathy is important to define at-risk patients, anticipate deterioration, and assess new therapies. ---------- RESEARCH DESIGN AND METHODS: A total of 101 diabetic patients and 17 age-matched control subjects underwent neurological evaluation, neurophysiology tests, quantitative sensory testing, and evaluation of corneal sensation and corneal nerve morphology using corneal confocal microscopy (CCM). ---------- RESULTS: Corneal sensation decreased significantly (P = 0.0001) with increasing neuropathic severity and correlated with the neuropathy disability score (NDS) (r = 0.441, P < 0.0001). Corneal nerve fiber density (NFD) (P < 0.0001), nerve fiber length (NFL), (P < 0.0001), and nerve branch density (NBD) (P < 0.0001) decreased significantly with increasing neuropathic severity and correlated with NDS (NFD r = −0.475, P < 0.0001; NBD r = −0.511, P < 0.0001; and NFL r = −0.581, P < 0.0001). NBD and NFL demonstrated a significant and progressive reduction with worsening heat pain thresholds (P = 0.01). Receiver operating characteristic curve analysis for the diagnosis of neuropathy (NDS >3) defined an NFD of <27.8/mm2 with a sensitivity of 0.82 (95% CI 0.68–0.92) and specificity of 0.52 (0.40–0.64) and for detecting patients at risk of foot ulceration (NDS >6) defined a NFD cutoff of <20.8/mm2 with a sensitivity of 0.71 (0.42–0.92) and specificity of 0.64 (0.54–0.74). ---------- CONCLUSIONS: CCM is a noninvasive clinical technique that may be used to detect early nerve damage and stratify diabetic patients with increasing neuropathic severity. Established diabetic neuropathy leads to pain and foot ulceration. Detecting neuropathy early may allow intervention with treatments to slow or reverse this condition (1). Recent studies suggested that small unmyelinated C-fibers are damaged early in diabetic neuropathy (2–4) but can only be detected using invasive procedures such as sural nerve biopsy (4,5) or skin-punch biopsy (6–8). Our studies have shown that corneal confocal microscopy (CCM) can identify early small nerve fiber damage and accurately quantify the severity of diabetic neuropathy (9–11). We have also shown that CCM relates to intraepidermal nerve fiber loss (12) and a reduction in corneal sensitivity (13) and detects early nerve fiber regeneration after pancreas transplantation (14). Recently we have also shown that CCM detects nerve fiber damage in patients with Fabry disease (15) and idiopathic small fiber neuropathy (16) when results of electrophysiology tests and quantitative sensory testing (QST) are normal. In this study we assessed corneal sensitivity and corneal nerve morphology using CCM in diabetic patients stratified for the severity of diabetic neuropathy using neurological evaluation, electrophysiology tests, and QST. This enabled us to compare CCM and corneal esthesiometry with established tests of diabetic neuropathy and define their sensitivity and specificity to detect diabetic patients with early neuropathy and those at risk of foot ulceration.

Immunohistochemical analysis of structural changes in collagen for the assessment of osteoarthritis

Collagen fibrillation within articular cartilage (AC) plays a key role in joint osteoarthritis (OA) progression and, therefore, studying collagen synthesis changes could be an indicator for use in the assessment of OA. Various staining techniques have been developed and used to determine the collagen network transformation under microscopy. However, because collagen and proteoglycan coexist and have the same index of refraction, conventional methods for specific visualization of collagen tissue is difficult. This study aimed to develop an advanced staining technique to distinguish collagen from proteoglycan and to determine its evolution in relation to OA progression using optical and laser scanning confocal microscopy (LSCM). A number of AC samples were obtained from sheep joints, including both healthy and abnormal joints with OA grades 1 to 3. The samples were stained using two different trichrome methods and immunohistochemistry (IHC) to stain both colourimetrically and with fluorescence. Using optical microscopy and LSCM, the present authors demonstrated that the IHC technique stains collagens only, allowing the collagen network to be separated and directly investigated. Fluorescently-stained IHC samples were also subjected to LSCM to obtain three-dimensional images of the collagen fibres. Changes in the collagen fibres were then correlated with the grade of OA in tissue. This study is the first to successfully utilize the IHC staining technique in conjunction with laser scanning confocal microscopy. This is a valuable tool for assessing changes to articular cartilage in OA.

Spatially offset Raman spectroscopy (SORS) for the analysis and detection of packaged pharmaceuticals and concealed drugs

Spatially offset Raman spectroscopy (SORS) is a powerful new technique for the non-invasive detection and identification of concealed substances and drugs. Here, we demonstrate the SORS technique in several scenarios that are relevant to customs screening, postal screening, drug detection and forensics applications. The examples include analysis of a multi-layered postal package to identify a concealed substance; identification of an antibiotic capsule inside its plastic blister pack; analysis of an envelope containing a powder; and identification of a drug dissolved in a clear solvent, contained in a non-transparent plastic bottle. As well as providing practical examples of SORS, the results highlight several considerations regarding the use of SORS in the field, including the advantages of different analysis geometries and the ability to tailor instrument parameters and optics to suit different types of packages and samples. We also discuss the features and benefits of SORS in relation to existing Raman techniques, including confocal microscopy, wide area illumination and the conventional backscattered Raman spectroscopy. The results will contribute to the recognition of SORS as a promising method for the rapid, chemically-specific analysis and detection of drugs and pharmaceuticals.

Analysis of micro-structural changes and measurement of their parameters of a food material

Food microstructure represents the way their elements arrangement and their interaction. Researchers in this field benefit from identifying new methods of examination of the microstructure and analysing the images. Experiments were undertaken to study micro-structural changes of food material during drying. Micro-structural images were obtained for potato samples of cubical shape at different moisture contents during drying using scanning electron microscopy. Physical parameters such as cell wall perimeter, and area were calculated using an image identification algorithm, based on edge detection and morphological operators. The algorithm was developed using Matlab.

Analyzing real-time video microscopy : the dynamics and geometry of vesicles and tubules in endocytosis

With the advent of live cell imaging microscopy, new types of mathematical analyses and measurements are possible. Many of the real-time movies of cellular processes are visually very compelling, but elementary analysis of changes over time of quantities such as surface area and volume often show that there is more to the data than meets the eye. This unit outlines a geometric modeling methodology and applies it to tubulation of vesicles during endocytosis. Using these principles, it has been possible to build better qualitative and quantitative understandings of the systems observed, as well as to make predictions about quantities such as ligand or solute concentration, vesicle pH, and membrane trafficked. The purpose is to outline a methodology for analyzing real-time movies that has led to a greater appreciation of the changes that are occurring during the time frame of the real-time video microscopy and how additional quantitative measurements allow for further hypotheses to be generated and tested.

Fast fourier analysis of structural organization in decellularized cartilage-on-bone laminates

Denaturation of tissues can provide a unique biological environment for regenerative medicine application only if minimal disruption of their microarchitecture is achieved during the decellularization process. The goal is to keep the structural integrity of such a construct as functional as the tissues from which they were derived. In this work, cartilage-on-bone laminates were decellularized through enzymatic, non-ionic and ionic protocols. This work investigated the effects of decellularization process on the microarchitecture of cartiligous extracellular matrix; determining the extent of how each process deteriorated the structural organization of the network. High resolution microscopy was used to capture cross-sectional images of samples prior to and after treatment. The variation of the microarchitecture was then analysed using a well defined fast Fourier image processing algorithm. Statistical analysis of the results revealed how significant the alternations among aforementioned protocols were (p < 0.05). Ranking the treatments by their effectiveness in disrupting the ECM integrity, they were ordered as: Trypsin> SDS> Triton X-100.

Anisotropy of articular cartilage reflects the ECM gradient architecture : Hough-Radon Transform Analysis

Tissue-specific extracellular matrix (ECM) is known to be an ideal bioscaffold to inspire the future of regenerative medicine. It holds the secret of how nature has developed such an organization of molecules into a unique functional complexity. This work exploited an innovative image processing algorithm and high resolution microscopy associated with mechanical analysis to establish a correlation between the gradient organization of cartiligous ECM and its anisotropic biomechanical response. This was hypothesized to be a reliable determinant that can elucidate how microarchitecture interrelates with biomechanical properties. Hough-Radon transform of the ECM cross-section images revealed its conformational variation from tangential interface down to subchondral region. As the orientation varied layer by layer, the anisotropic mechanical response deviated relatively. Although, results were in good agreement (Kendall's tau-b > 90%), there were evidences proposing that alignment of the fibrous network, specifically in middle zone, is not as random as it was previously thought.

Utility of corneal confocal microscopy for assessing mild diabetic neuropathy: baseline findings of the LANDMark study

Background:  For those in the field of managing diabetic complications, the accurate diagnosis and monitoring of diabetic peripheral neuropathy (DPN) continues to be a challenge. Assessment of sub-basal corneal nerve morphology has recently shown promise as a novel ophthalmic marker for the detection of DPN. Methods:  Two hundred and thirty-one individuals with diabetes with predominantly mild or no neuropathy and 61 controls underwent evaluation of diabetic neuropathy symptom score, neuropathy disability score, testing with 10 g monofilament, quantitative sensory testing (warm, cold, vibration detection) and nerve conduction studies. Corneal nerve fibre length, branch density and tortuosity were measured using corneal confocal microscopy. Differences in corneal nerve morphology between individuals with and without DPN and controls were investigated using analysis of variance and correlations were determined between corneal morphology and established tests of, and risk factors for, DPN. Results:  Corneal nerve fibre length was significantly reduced in diabetic individuals with mild DPN compared with both controls (p < 0.001) and diabetic individuals without DPN (p = 0.012). Corneal nerve branch density was significantly reduced in individuals with mild DPN compared with controls (p = 0.032). Corneal nerve fibre tortuosity did not show significant differences. Corneal nerve fibre length and corneal nerve branch density showed modest correlations to most measures of neuropathy, with the strongest correlations to nerve conduction study parameters (r = 0.15 to 0.25). Corneal nerve fibre tortuosity showed only a weak correlation to the vibration detection threshold. Corneal nerve fibre length was inversely correlated to glycated haemoglobin (r = -0.24) and duration of diabetes (r = -0.20). Conclusion:  Assessment of corneal nerve morphology is a non-invasive, rapid test capable of showing differences between individuals with and without DPN. Corneal nerve fibre length shows the strongest associations with other diagnostic tests of neuropathy and with established risk factors for neuropathy.

Automatic particle picking of biological molecules imaged by electron microscopy

One of the next great challenges of cell biology is the determination of the enormous number of protein structures encoded in genomes. In recent years, advances in electron cryo-microscopy and high-resolution single particle analysis have developed to the point where they now provide a methodology for high resolution structure determination. Using this approach, images of randomly oriented single particles are aligned computationally to reconstruct 3-D structures of proteins and even whole viruses. One of the limiting factors in obtaining high-resolution reconstructions is obtaining a large enough representative dataset ($>100,000$ particles). Traditionally particles have been manually picked which is an extremely labour intensive process. The problem is made especially difficult by the low signal-to-noise ratio of the images. This paper describes the development of automatic particle picking software, which has been tested with both negatively stained and cryo-electron micrographs. This algorithm has been shown to be capable of selecting most of the particles, with few false positives. Further work will involve extending the software to detect differently shaped and oriented particles.

Analytical electron microscopy of Mg-SiO smokes; a comparison with infrared and XRD studies

Experimentally obtained Mg.SiO smokes were studied by analytical electron microscopy using the same samples that had been previously characterized by repeated infrared spectroscopy. Analytical electron microscopy shows that unannealed smokes contain some degree of microcrystallinity which increases with increased annealing for up to 30 hr. An SiO2 polymorph (tridymite) and MgO may form contemporaneously as a result of growth of forsterite (Mg2SiO4) microcrystallites in the initially nonstoichiometric smokes. After 4 hr annealing, forsterite and tridymite react to enstatite (MgSiO3). We suggest that infrared spectroscopy and X-ray diffraction analysis should be complemented by detailed analytical electron microscopy to detect budding crystallinity in vapor phase condensates.

Porohyperelastic analysis to explore mechanical properties of chondrocytes using numerical modeling and experiments : a finite element study

There are many continuum mechanical models have been developed such as liquid drop models, solid models, and so on for single living cell biomechanics studies. However, these models do not give a fully approach to exhibit a clear understanding of the behaviour of single living cells such as swelling behaviour, drag effect, etc. Hence, the porohyperelastic (PHE) model which can capture those aspects would be a good candidature to study cells behaviour (e.g. chondrocytes in this study). In this research, an FEM model of single chondrocyte cell will be developed by using this PHE model to simulate Atomic Force Microscopy (AFM) experimental results with the variation of strain rate. This material model will be compared with viscoelastic model to demonstrate the advantages of PHE model. The results have shown that the maximum value of force applied of PHE model is lower at lower strain rates. This is because the mobile fluid does not have enough time to exude in case of very high strain rate and also due to the lower permeability of the membrane than that of the protoplasm of chondrocyte. This behavior is barely observed in viscoelastic model. Thus, PHE model is the better model for cell biomechanics studies.

Electron microscopy of micron-sized lunar soil

We have performed electron-microscopic analysis on 0.5-1.0µm grains in order to study radiation damage by the solar-wind. We are reporting some interesting results we have found in monomineralic grains from core sample 15010,1130. This is a submature soil which has been studied for rare gas abundance and ferromagnetic resonance by (1) and modal petrology by (2).

Porohyperelastic analysis of single osteocyte using AFM and inverse FEA

Osteocytes are the mature cells and perform as mechanosensors within the bone. The mechanical property of osteocytes plays an important role to fulfill these functions. However, little researches have been done to investigate the mechanical deformation properties of single osteocytes. Atomic Force Microscopy (AFM) is a state-of-art experimental facility for high resolution imaging of tissues, cells and any surfaces as well as for probing mechanical properties of the samples both qualitatively and quantitatively. In this paper, the experimental study based on AFM is firstly used to obtain forceindentation curves of single round osteocytes. The porohyperelastic (PHE) model of a single osteocyte is then developed by using the inverse finite element analysis (FEA) to identify and extract mechanical properties from the experiment results. It has been found that the PHE model is a good candidature for biomechanics studies of osteocytes.