A solvent-free matrix application method for matrix-assisted laser desorption/ionization imaging of small molecules

Matrix application continues to be a critical step in sample preparation for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI). Imaging of small molecules such as drugs and metabolites is particularly problematic because the commonly used washing steps to remove salts are usually omitted as they may also remove the analyte, and analyte spreading is more likely with conventional wet matrix application methods. We have developed a method which uses the application of matrix as a dry, finely divided powder, here referred to as dry matrix application, for the imaging of drug compounds. This appears to offer a complementary method to wet matrix application for the MALDI-MSI of small molecules, with the alternative matrix application techniques producing different ion profiles, and allows the visualization of compounds not observed using wet matrix application methods. We demonstrate its value in imaging clozapine from rat kidney and 4-bromophenyl-1,4-diazabicyclo(3.2.2)nonane-4-carboxylic acid from rat brain. In addition, exposure of the dry matrix coated sample to a saturated moist atmosphere appears to enhance the visualization of a different set of molecules.

Comparison of techniques for measuring anterior chamber depth:Orbscan imaging, Smith's technique, and van Herick's method

Background Evaluation of anterior chamber depth (ACD) can potentially identify those patients at risk of angle-closure glaucoma. We aimed to: compare van Herick’s limbal chamber depth (LCDvh) grades with LCDorb grades calculated from the Orbscan anterior chamber angle values; determine Smith’s technique ACD and compare to Orbscan ACD; and calculate a constant for Smith’s technique using Orbscan ACD. Methods Eighty participants free from eye disease underwent LCDvh grading, Smith’s technique ACD, and Orbscan anterior chamber angle and ACD measurement. Results LCDvh overestimated grades by a mean of 0.25 (coefficient of repeatability [CR] 1.59) compared to LCDorb. Smith’s technique (constant 1.40 and 1.31) overestimated ACD by a mean of 0.33 mm (CR 0.82) and 0.12 mm (CR 0.79) respectively, compared to Orbscan. Using linear regression, we determined a constant of 1.22 for Smith’s slit-length method. Conclusions Smith’s technique (constant 1.31) provided an ACD that is closer to that found with Orbscan compared to a constant of 1.40 or LCDvh. Our findings also suggest that Smith’s technique would produce values closer to that obtained with Orbscan by using a constant of 1.22.

Attenuated total reflection-FT-IR spectroscopic imaging of protein crystallization

Protein crystallization is of strategic and commercial relevance in the post-genomic era because of its pivotal role in structural proteomics projects. Although protein structures are crucial for understanding the function of proteins and to the success of rational drug design and other biotechnology applications, obtaining high quality crystals is a major bottleneck to progress. The major means of obtaining crystals is by massive-scale screening of a target protein solution with numerous crystallizing agents. However, when crystals appear in these screens, one cannot easily know if they are crystals of protein, salt, or any other molecule that happens to be present in the trials. We present here a method based on Attenuated Total Reflection (ATR)-FT-IR imaging that reliably identifies protein crystals through a combination of chemical specificity and the visualizing capability of this approach, thus solving a major hurdle in protein crystallization. ATR-FT-IR imaging was successfully applied to study the crystallization of thaumatin and lysozyme in a high-throughput manner, simultaneously from six different solutions. This approach is fast as it studies protein crystallization in situ and provides an opportunity to examine many different samples under a range of conditions.

Use of fundus imaging in quantification of age-related macular change

This review will discuss the use of manual grading scales, digital photography, and automated image analysis in the quantification of fundus changes caused by age-related macular disease. Digital imaging permits processing of images for enhancement, comparison, and feature quantification, and these techniques have been investigated for automated drusen analysis. The accuracy of automated analysis systems has been enhanced by the incorporation of interactive elements, such that the user is able to adjust the sensitivity of the system, or manually add and remove pixels. These methods capitalize on both computer and human image feature recognition and the advantage of computer-based methodologies for quantification. The histogram-based adaptive local thresholding system is able to extract useful information from the image without being affected by the presence of other structures. More recent developments involve compensation for fundus background reflectance, which has most recently been combined with the Otsu method of global thresholding. This method is reported to provide results comparable with manual stereo viewing. Developments in this area are likely to encourage wider use of automated techniques. This will make the grading of photographs easier and cheaper for clinicians and researchers. © 2007 Elsevier Inc. All rights reserved.

A general linear model for MEG beamformer imaging

A new general linear model (GLM) beamformer method is described for processing magnetoencephalography (MEG) data. A standard nonlinear beamformer is used to determine the time course of neuronal activation for each point in a predefined source space. A Hilbert transform gives the envelope of oscillatory activity at each location in any chosen frequency band (not necessary in the case of sustained (DC) fields), enabling the general linear model to be applied and a volumetric T statistic image to be determined. The new method is illustrated by a two-source simulation (sustained field and 20 Hz) and is shown to provide accurate localization. The method is also shown to locate accurately the increasing and decreasing gamma activities to the temporal and frontal lobes, respectively, in the case of a scintillating scotoma. The new method brings the advantages of the general linear model to the analysis of MEG data and should prove useful for the localization of changing patterns of activity across all frequency ranges including DC (sustained fields). © 2004 Elsevier Inc. All rights reserved.

Spatio-temporal imaging of corticaldesynchronization in migraine visual aura:a magnetoencephalography case study

Objective.-To determine cortical oscillatory changes involved in migraine visual aura using magnetoencephalography (MEG). Background.-Visual aura in the form of scintillating scotoma precedes migraine in many cases. The involvement of cortical spreading depression within striate and extra-striate cortical areas is implicated in the generation of the disturbance, but the details of its progression, the effects on cortical oscillations, and the mechanisms of aura generation are unclear. Methods.-We used MEG to directly image changes in cortical oscillatory power during an episode of scintillating scotoma in a patient who experiences aura without subsequent migraine headache. Using the synthetic aperture magnetometry method of MEG source imaging, focal changes in cortical oscillatory power were observed over a 20-minute period and visualized in coregistration with the patient's magnetic resonance image. Results.-Alpha band desynchronization in both the left extra-striate and temporal cortex persisted for the duration of reported visual disturbance, terminating abruptly upon disappearance of scintillations. Gamma frequency desynchronization in the left temporal lobe continued for 8 to 10 minutes following the reported end of aura. Conclusions.-Observations implicate the extra-striate and temporal cortex in migraine visual aura and suggest involvement of alpha desynchronization in generation of phosphenes and gamma desynchronization in sustained inhibition of visual function.

A novel method for studying the cortical processing of human swallowing using Synthetic Aperture Magnetometry(SAM)

Background/Aims: Positron emission tomography has been applied to study cortical activation during human swallowing, but employs radio-isotopes precluding repeated experiments and has to be performed supine, making the task of swallowing difficult. Here we now describe Synthetic Aperture Magnetometry (SAM) as a novel method of localising and imaging the brain's neuronal activity from magnetoencephalographic (MEG) signals to study the cortical processing of human volitional swallowing in the more physiological prone position. Methods: In 3 healthy male volunteers (age 28–36), 151-channel whole cortex MEG (Omega-151, CTF Systems Inc.) was recorded whilst seated during the conditions of repeated volitional wet swallowing (5mls boluses at 0.2Hz) or rest. SAM analysis was then performed using varying spatial filters (5–60Hz) before co-registration with individual MRI brain images. Activation areas were then identified using standard sterotactic space neuro-anatomical maps. In one subject repeat studies were performed to confirm the initial study findings. Results: In all subjects, cortical activation maps for swallowing could be generated using SAM, the strongest activations being seen with 10–20Hz filter settings. The main cortical activations associated with swallowing were in: sensorimotor cortex (BA 3,4), insular cortex and lateral premotor cortex (BA 6,8). Of relevance, each cortical region displayed consistent inter-hemispheric asymmetry, to one or other hemisphere, this being different for each region and for each subject. Intra-subject comparisons of activation localisation and asymmetry showed impressive reproducibility. Conclusion: SAM analysis using MEG is an accurate, repeatable, and reproducible method for studying the brain processing of human swallowing in a more physiological manner and provides novel opportunities for future studies of the brain-gut axis in health and disease.

Three-dimensional modeling of the human eye based on magnetic resonance imaging

PURPOSE. A methodology for noninvasively characterizing the three-dimensional (3-D) shape of the complete human eye is not currently available for research into ocular diseases that have a structural substrate, such as myopia. A novel application of a magnetic resonance imaging (MRI) acquisition and analysis technique is presented that, for the first time, allows the 3-D shape of the eye to be investigated fully. METHODS. The technique involves the acquisition of a T2-weighted MRI, which is optimized to reveal the fluid-filled chambers of the eye. Automatic segmentation and meshing algorithms generate a 3-D surface model, which can be shaded with morphologic parameters such as distance from the posterior corneal pole and deviation from sphericity. Full details of the method are illustrated with data from 14 eyes of seven individuals. The spatial accuracy of the calculated models is demonstrated by comparing the MRI-derived axial lengths with values measured in the same eyes using interferometry. RESULTS. The color-coded eye models showed substantial variation in the absolute size of the 14 eyes. Variations in the sphericity of the eyes were also evident, with some appearing approximately spherical whereas others were clearly oblate and one was slightly prolate. Nasal-temporal asymmetries were noted in some subjects. CONCLUSIONS. The MRI acquisition and analysis technique allows a novel way of examining 3-D ocular shape. The ability to stratify and analyze eye shape, ocular volume, and sphericity will further extend the understanding of which specific biometric parameters predispose emmetropic children subsequently to develop myopia. Copyright © Association for Research in Vision and Ophthalmology.

Use of a solvent-free dry matrix coating for quantitative matrix-assisted laser desorption ionization imaging of 4-bromophenyl-1,4-diazabicyclo(3.2.2)nonane-4-carboxylate in rat brain and quantitative analysis of the drug from laser microdissected tissue regions

A dry matrix application for matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) was used to profile the distribution of 4-bromophenyl-1,4-diazabicyclo(3.2.2)nonane-4-carboxylate, monohydrochloride (BDNC, SSR180711) in rat brain tissue sections. Matrix application involved applying layers of finely ground dry alpha-cyano-4-hydroxycinnamic acid (CHCA) to the surface of tissue sections thaw mounted onto MALDI targets. It was not possible to detect the drug when applying matrix in a standard aqueous-organic solvent solution. The drug was detected at higher concentrations in specific regions of the brain, particularly the white matter of the cerebellum. Pseudomultiple reaction monitoring imaging was used to validate that the observed distribution was the target compound. The semiquantitative data obtained from signal intensities in the imaging was confirmed by laser microdissection of specific regions of the brain directed by the imaging, followed by hydrophilic interaction chromatography in combination with a quantitative high-resolution mass spectrometry method. This study illustrates that a dry matrix coating is a valuable and complementary matrix application method for analysis of small polar drugs and metabolites that can be used for semiquantitative analysis.

Coherence imaging by use of a Newton rings sampling function

We show that, with suitable optics in the arm of a Michelson interferometer, orthogonal galvo-scanning mirrors build a sampling function in the form of Newton rings when the two interferometer arms are matched. Using a low-coherence source, one can obtain transversal depth-resolved images. A fast display procedure using a storage oscilloscope was devised based on this method.

Multispectral retinal image analysis:a novel non-invasive tool for retinal imaging

Purpose-To develop a non-invasive method for quantification of blood and pigment distributions across the posterior pole of the fundus from multispectral images using a computer-generated reflectance model of the fundus. Methods - A computer model was developed to simulate light interaction with the fundus at different wavelengths. The distribution of macular pigment (MP) and retinal haemoglobins in the fundus was obtained by comparing the model predictions with multispectral image data at each pixel. Fundus images were acquired from 16 healthy subjects from various ethnic backgrounds and parametric maps showing the distribution of MP and of retinal haemoglobins throughout the posterior pole were computed. Results - The relative distributions of MP and retinal haemoglobins in the subjects were successfully derived from multispectral images acquired at wavelengths 507, 525, 552, 585, 596, and 611?nm, providing certain conditions were met and eye movement between exposures was minimal. Recovery of other fundus pigments was not feasible and further development of the imaging technique and refinement of the software are necessary to understand the full potential of multispectral retinal image analysis. Conclusion - The distributions of MP and retinal haemoglobins obtained in this preliminary investigation are in good agreement with published data on normal subjects. The ongoing development of the imaging system should allow for absolute parameter values to be computed. A further study will investigate subjects with known pathologies to determine the effectiveness of the method as a screening and diagnostic tool.

Drusen detection in retro-mode imaging by a scanning laser ophthalmoscope

Purpose: The Nidek F-10 is a scanning laser ophthalmoscope that is capable of a novel fundus imaging technique, so-called ‘retro-mode’ imaging. The standard method of imaging drusen in age-related macular degeneration (AMD) is by fundus photography. The aim of the study was to assess drusen quantification using retro-mode imaging. Methods: Stereoscopic fundus photographs and retro-mode images were captured in 31 eyes of 20 patients with varying stages of AMD. Two experienced masked retinal graders independently assessed images for the number and size of drusen, using purpose-designed software. Drusen were further assessed in a subset of eight patients using optical coherence tomography (OCT) imaging. Results: Drusen observed by fundus photography (mean 33.5) were significantly fewer in number than subretinal deposits seen in retro-mode (mean 81.6; p < 0.001). The predominant deposit diameter was on average 5 µm smaller in retro-mode imaging than in fundus photography (p = 0.004). Agreement between graders for both types of imaging was substantial for number of deposits (weighted ? = 0.69) and moderate for size of deposits (weighted ? = 0.42). Retro-mode deposits corresponded to drusen on OCT imaging in all eight patients. Conclusion: The subretinal deposits detected by retro-mode imaging were consistent with the appearance of drusen on OCT imaging; however, a larger longitudinal study would be required to confirm this finding. Retro-mode imaging detected significantly more deposits than conventional colour fundus photography. Retro-mode imaging provides a rapid non-invasive technique, useful in monitoring subtle changes and progression of AMD, which may be useful in monitoring the response of drusen to future therapeutic interventions.

Retinotopic mapping of the primary visual cortex - a challenge for MEG imaging of the human cortex

Magnetoencephalography (MEG) can be used to reconstruct neuronal activity with high spatial and temporal resolution. However, this reconstruction problem is ill-posed, and requires the use of prior constraints in order to produce a unique solution. At present there are a multitude of inversion algorithms, each employing different assumptions, but one major problem when comparing the accuracy of these different approaches is that often the true underlying electrical state of the brain is unknown. In this study, we explore one paradigm, retinotopic mapping in the primary visual cortex (V1), for which the ground truth is known to a reasonable degree of accuracy, enabling the comparison of MEG source reconstructions with the true electrical state of the brain. Specifically, we attempted to localize, using a beanforming method, the induced responses in the visual cortex generated by a high contrast, retinotopically varying stimulus. Although well described in primate studies, it has been an open question whether the induced gamma power in humans due to high contrast gratings derives from V1 rather than the prestriate cortex (V2). We show that the beanformer source estimate in the gamma and theta bands does vary in a manner consistent with the known retinotopy of V1. However, these peak locations, although retinotopically organized, did not accurately localize to the cortical surface. We considered possible causes for this discrepancy and suggest that improved MEG/magnetic resonance imaging co-registration and the use of more accurate source models that take into account the spatial extent and shape of the active cortex may, in future, improve the accuracy of the source reconstructions.

In situ imaging and height reconstruction of phase separation processes in polymer blends during spin coating

Spin coating polymer blend thin films provides a method to produce multiphase functional layers of high uniformity covering large surface areas. Applications for such layers include photovoltaics and light-emitting diodes where performance relies upon the nanoscale phase separation morphology of the spun film. Furthermore, at micrometer scales, phase separation provides a route to produce self-organized structures for templating applications. Understanding the factors that determine the final phase-separated morphology in these systems is consequently an important goal. However, it has to date proved problematic to fully test theoretical models for phase separation during spin coating, due to the high spin speeds, which has limited the spatial resolution of experimental data obtained during the coating process. Without this fundamental understanding, production of optimized micro- and nanoscale structures is hampered. Here, we have employed synchronized stroboscopic illumination together with the high light gathering sensitivity of an electron-multiplying charge-coupled device camera to optically observe structure evolution in such blends during spin coating. Furthermore the use of monochromatic illumination has allowed interference reconstruction of three-dimensional topographies of the spin-coated film as it dries and phase separates with nanometer precision. We have used this new method to directly observe the phase separation process during spinning for a polymer blend (PS-PI) for the first time, providing new insights into the spin-coating process and opening up a route to understand and control phase separation structures. © 2011 American Chemical Society.

Three-dimensional fluid pressure mapping in porous media using magnetic resonance imaging with gas-filled liposomes

This paper presents and demonstrates a method for using magnetic resonance imaging to measure local pressure of a fluid saturating a porous medium. The method is tested both in a static system of packed silica gel and in saturated sintered glass cylinders experiencing fluid flow. The fluid used contains 3% gas in the form of 3-μm average diameter gas filled 1,2-distearoyl-sn-glycero-3-phosphocholine (C18:0, MW: 790.16) liposomes suspended in 5% glycerol and 0.5% Methyl cellulose with water. Preliminary studies at 2.35 T demonstrate relative magnetic resonance signal changes of 20% per bar in bulk fluid for an echo time TE=40 ms, and 6-10% in consolidated porous media for TE=10 ms, over the range 0.8-1.8 bar for a spatial resolution of 0.1 mm3 and a temporal resolution of 30 s. The stability of this solution with relation to applied pressure and methods for improving sensitivity are discussed. © 2007 Elsevier Inc. All rights reserved.