Microscopic phase reconstruction of cervical exfoliated cell under partially coherent illumination.

Basic coherent diffraction imaging methods strongly rely on having a highly coherent illumination in order to reconstruct the phase accurately. However, regardless of considering the turbulent transport medium, the instability of the system or the generation mechanism of the light source, partially coherent illumination is more common in real case. In this paper, we proposed an efficient microscopic phase imaging method to study normal and abnormal cervical exfoliated cells. By applying three phase modulations in a single point of the sample's transmitted field, the phase can be retrieved with correspoding three intensities under partially coherent illumination. Compared with intensity map, we can efficiently and clearly judge the proportion of high density shrinking abnormal cells from the phase distributions, which provides a confident analysis and evaluation basis for early medical diagnosis of cervical cancer. This study also has potential applications in noninvasive optical imaging of dynamic biological tissues.

Phase retrieval algorithms for lensless imaging using diffractive shearing interferometry.

Diffractive shearing interferometry (DSI) is a method that has recently been developed to perform lensless imaging using extreme ultraviolet radiation generated by high-harmonic generation. In this paper, we investigate the uniqueness of the DSI solution and the requirements for the support constraint size. We find that there can be multiple solutions to the DSI problem that consist of displaced copies of the actual object. These alternative solutions can be eliminated by enforcing a sufficiently tight support constraint, or by introducing additional synthetic constraints. We furthermore propose a new DSI algorithm inspired by the analogy with coherent diffractive imaging (CDI) algorithms: the original DSI algorithm is in a way analogous to the hybrid input-output algorithm as used in CDI, and we propose a new algorithm that is more analogous to the error reduction algorithm as used in CDI. We find that the newly proposed algorithm is suitable for final refinement of the reconstruction.

Extreme ultraviolet lensless imaging without object support through rotational diversity in diffractive shearing interferometry.

We report on a method that allows microscopic image reconstruction from extreme-ultraviolet diffraction patterns without the need for object support constraints or other prior knowledge about the object structure. This is achieved by introducing additional diversity through rotation of an object in a rotationally asymmetric probe beam, produced by the spatial interference between two phase-coherent high-harmonic beams. With this rotational diffractive shearing interferometry method, we demonstrate robust image reconstruction of microscopic objects at wavelengths around 30 nm, using images recorded at only three to five different object rotations.

Angular momentum properties of hybrid cylindrical vector vortex beams in tightly focused optical systems.

Optical angular momenta (AM) have attracted tremendous research interest in recent years. In this paper we theoretically investigate the electromagnetic field and angular momentum properties of tightly focused arbitrary cylindrical vortex vector (CVV) input beams. An absorptive particle is placed in focused CVV fields to analyze the optical torques. The spin-orbit motions of the particle can be predicted and controlled when the influences of different parameters, such as the topological charge, the polarization and the initial phases, are taken into account. These findings will be helpful in optical beam shaping, optical spin-orbit interaction and practical optical manipulation.

Lateral position correction in ptychography using the gradient of intensity patterns.

Ptychography, a form of Coherent Diffractive Imaging, is used with short wavelengths (e.g. X-rays, electron beams) to achieve high-resolution image reconstructions. One of the limiting factors for the reconstruction quality is the accurate knowledge of the illumination probe positions. Recently, many advances have been made to relax the requirement for the probe positions accuracy. Here, we analyse and demonstrate a straightforward approach that can be used to correct the probe positions with sub-pixel accuracy. Simulations and experimental results with visible light are presented in this work.

Non-iterative method for phase retrieval and coherence characterization by focus variation using a fixed star-shaped mask.

A novel non-iterative phase retrieval method is proposed and demonstrated with a proof-of-principle experiment. The method uses a fixed specially designed mask and through-focus intensity measurements. It is demonstrated that this method is robust to spatial partial coherence in the illumination, making it suitable for coherent diffractive imaging using spatially partially coherent light, as well as for coherence characterization.

Model-independent noise-robust extension of ptychography.

A noise-robust extension of iterative phase retrieval algorithms that does not need to assume a noise model is proposed. It works by adapting the intensity constraints using the reconstructed object. Using a proof-of-principle ptychographic experiment with visible light and a spatial light modulator to create an object, the proposed method is tested and it compares favorably to the Extended Ptychographic Iterative Engine (ePIE) with reduced step size. The method is general, so it can also be applied to other iterative reconstruction schemes such as phase retrieval using focus variation.

Phase retrieval of the full vectorial field applied to coherent Fourier scatterometry.

Coherent Fourier scatterometry is an optical metrology technique that utilizes the measured intensity of the scattered optical field to reconstruct certain parameters of test structures written on a wafer with nano-scale accuracy. The intensity of the scattered field is recorded with a camera and this information is used to retrieve the grating parameters. To improve sensitivity in the parameter reconstruction, the phase of the scattered field can also be acquired. Interferometry can be used for this purpose, but with the cost of cumbersomeness. In this paper, we show that iterative phase retrieval methods can be applied to retrieve the scattered complex fields from only intensity measurement data. We show that the accuracy of the retrieved complex fields using phase retrieval is comparable to that measured directly using interferometry.

Non-iterative phase retrieval by phase modulation through a single parameter.

We report on a novel non-iterative phase retrieval method with which the complex-valued transmission function of an object can be retrieved with a non-iterative computation, with a limited number of intensity measurements. The measurements are taken in either real space or Fourier space, and for each measurement the phase in its dual space is modulated according to a single optical parameter. The requirement found for the phase modulation function is a general one, which therefore allows for plenty of customization in this method. It is shown that quantitative Zernike phase contrast imaging is one special case of this general method. With simulations we investigate the sampling requirements for a microscopy setup and for a Coherent Diffraction Imaging (CDI) setup.

Combining ptychographical algorithms with the Hybrid Input-Output (HIO) algorithm.

In this article we combine the well-known Ptychographical Iterative Engine (PIE) with the Hybrid Input-Output (HIO) algorithm. The important insight is that the HIO feedback function should be kept strictly separate from the reconstructed object, which is done by introducing a separate feedback function per probe position. We have also combined HIO with floating PIE (fPIE) and extended PIE (ePIE). Simulations indicate that the combined algorithm performs significantly better in many situations. Although we have limited our research to a combination with HIO, the same insight can be used to combine ptychographical algorithms with any phase retrieval algorithm that uses a feedback function.

Demonstration of an optimised focal field with long focal depth and high transmission obtained with the Extended Nijboer-Zernike theory.

In several optical systems, a specific Point Spread Function (PSF) needs to be generated. This can be achieved by shaping the complex field at the pupil. The Extended Nijboer-Zernike (ENZ) theory relates complex Zernike modes on the pupil directly to functions in the focal region. In this paper, we introduce a method to engineer a PSF using the ENZ theory. In particular, we present an optimization algorithm to design an extended depth of focus with high lateral resolution, while keeping the transmission of light high (over 60%). We also have demonstrated three outcomes of the algorithm using a Spatial Light Modulator (SLM).

KCNJ10 mutations display differential sensitivity to heteromerisation with KCNJ16.

BACKGROUND/AIMS: Mutations in the inwardly-rectifying K(+)-channel KCNJ10/Kir4.1 cause autosomal recessive EAST syndrome (epilepsy, ataxia, sensorineural deafness and tubulopathy). KCNJ10 is expressed in the distal convoluted tubule of the kidney, stria vascularis of the inner ear and brain glial cells. Patients diagnosed clinically with EAST syndrome were genotyped and mutations in KCNJ10 were studied functionally. METHODS: Patient DNA was amplified and sequenced, and new mutations were identified. Mutant and wild-type KCNJ10 constructs were cloned and heterologously expressed in Xenopus oocytes. Whole-cell K(+) currents were measured by 2-electrode voltage clamping and channel expression was analysed by Western blotting. RESULTS: We identified 3 homozygous mutations in KCNJ10 (p.F75C, p.A167V and p.V91fs197X), with mutation p.A167V previously reported in a compound heterozygous state. Oocytes expressing wild-type human KCNJ10 showed inwardly rectified currents, which were significantly reduced in all of the mutants (p < 0.001). Specific inhibition of KCNJ10 currents by Ba(2+) demonstrated a large residual function in p.A167V only, which was not compatible with causing disease. However, co-expression with KCNJ16 abolished function in these heteromeric channels almost completely. CONCLUSION: This study provides an explanation for the pathophysiology of the p.A167V KCNJ10 mutation, which had previously not been considered pathogenic on its own. These findings provide evidence for the functional cooperation of KCNJ10 and KCNJ16. Thus, in vitro ascertainment of KCNJ10 function may necessitate co-expression with KCNJ16.

Native ion mobility-mass spectrometry and related methods in structural biology.

Mass spectrometry-based methods have become increasingly important in structural biology - in particular for large and dynamic, even heterogeneous assemblies of biomolecules. Native electrospray ionization coupled to ion mobility-mass spectrometry provides access to stoichiometry, size and architecture of noncovalent assemblies; while non-native approaches such as covalent labeling and H/D exchange can highlight dynamic details of protein structures and capture intermediate states. In this overview article we will describe these methods and highlight some recent applications for proteins and protein complexes, with particular emphasis on native MS analysis. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.

[No relationship between so-called 'haemopyrrollactamuria' and fatigue in secondary-school students]. / Geen verband tussen zogeheten 'hemopyrrollactamurie' en vermoeidheid bij gymnasiasten.

OBJECTIVE: To determine the relationship between urinary haemopyrrollactam (HPL) and subjective fatigue in secondary-school students. DESIGN: Cross-sectional and descriptive. METHOD: Urine samples were collected from 75 secondary-school students (43 boys and 32 girls, aged 16-18 years) in Nijmegen, the Netherlands. The samples were tested for HPL complex at the Centre for Environmental Medicine (Klinisch Ecologisch Allergie Centrum; KEAC) in Weert. Students also completed the 'Abbreviated fatigue questionnaire'. A score of 21 points was considered the threshold for fatigue. RESULTS: Of the 75 participants, 22 (29%) had increased HPL complex excretion, including 15 boys (35%) and 7 girls (22%). Overall, 19 (25%) had a fatigue score > 21: 16 boys (37%) and 3 girls (9%). There was no statistical relationship between increased HPL complex excretion and fatigue score. CONCLUSION: These findings do not support a relationship between fatigue and increased urinary HPL complex excretion.

Children with unexplained chronic pain: substantial impairment in everyday life.

AIMS: To describe and quantify impairment in an outpatient population of children with chronic pain of unknown origin (UCP). METHODS: A total of 149 children who presented with pain of at least three months' duration and without a satisfactory explanation at presentation were studied. Number of somatic complaints (Children's Somatisation Inventory, CSI), pain intensity (VAS, 0-10 cm), functional disability (Child Health Questionnaire (CHQ-CF) and clinical history), and general health perceptions (CHQ) were determined. RESULTS: Mean age of the children was 11.8 years; 73% were girls. Overall, 72% suffered impairment in sports activities, 51% reported absence from school, 40% experienced limitations in social functioning, and 34% had problems with sleeping. Mean number of somatic symptoms differed significantly between boys (8.4) and girls (10.7). The CHQ-CF scores for physical functioning, role/social functioning, and general health perceptions were 76.4, 70.7, and 57.5, respectively, indicating substantial impairment on all domains. The mean pain intensity was 4.7 for current and 7.1 for worst pain. Children solely evaluated by a general practitioner prior to referral reported less, though still substantial, impairment. Low general health perceptions, impaired role/social functioning, high pain intensity, and having headache or musculoskeletal pain were independent predictors of having significant impairment. CONCLUSIONS: Referred children with UCP show substantial impairment on multiple domains in daily life.

Intracellular and extracellular, ionized and total magnesium in pre-eclampsia and uncomplicated pregnancy.

Magnesium (Mg) and calcium (CA) concentrations in women with pre-eclampsia, women with an uncomplicated pregnancy and non-pregnant women were compared. Ionized serum magnesium and calcium concentrations and intracellular magnesium concentrations were measured in 15 pregnant women with severe pre-eclampsia, 34 uncomplicated pregnant women early, at midterm and preterm in their pregnancy and 24 non-pregnant women. The ionized calcium concentration did not chance during normal pregnancy or during pre-eclampsia relative to non-pregnant women. In contrast, elevated total and ionized magnesium serum concentrations were found in women with severe pre-eclampsia (total Mg = 0.85+/-0.11 mM, ionized Mg = 0.61+/-0.06 mM) relative to uncomplicated pregnant women (total Mg = 0.72+/-0.06 mM, ionized Mg = 0.53+/-0.03 mM). Total magnesium in pre-eclamptic women were similar to non-pregnant women. Intracellular ionized and total magnesium concentrations in mononuclear blood cells and erythrocytes were similar in pre-eclamptic women and women with uncomplicated pregnancy. Serum magnesium concentrations are elevated in severe pre-eclamptic women relative to women with uncomplicated pregnancy and are related to birth weight and gestational age at delivery. There may be a causal relationship since magnesium is involved in blood pressure regulation through an intracellular inhibition of NO synthase in endothelial cells.

[Preeclampsia not (yet) predictable from the blood platelet count]. / Preëclampsie (nog) niet te voorspellen door metingen aan de bloedplaatjes.

Platelet activation plays an important role in the pathogenesis of pre-eclampsia as demonstrated by reduced platelet count, increased mean platelet volume and elevated plasma concentrations of beta-thromboglobulin and platelet factor 4 in pre-eclamptic patients. Thrombocytopenia precedes the onset of clinical symptoms of pre-eclampsia. Possibly an enhanced activation state of the platelets in the circulation is present some time before the onset of pre-eclampsia: platelet activation might then be used to predict pre-eclampsia. Flow cytometry is considered the most sensitive technique at this moment to measure the activation of platelets. Fluorescent-labelled antibodies are used to detect antigens that appear on the platelet surface or change their conformation upon activation. During the first and second trimester of pregnancy pre-eclamptic patients have an increased expression of some antigens on the surface of their platelets, such as CD63. There is no reliable platelet test yet to predict the onset of pre-eclampsia.

Lorenzo's oil and platelet activation in adrenomyeloneuropathy and asymptomatic X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (X-ALD) is an inherited disorder of peroxisomal beta-oxidation, which results in accumulation of very long-chain fatty acids, causing damage to the nervous system, adrenal cortex and testis. The two most frequent phenotypes are childhood cerebral adrenoleukodystrophy (CCALD) and adrenomyeloneuropathy (AMN). Some affected males demonstrate no clinical signs (asymptomatic ALD), whereas female carriers can also be affected. Patients with X-ALD have been treated with Lorenzo's oil, a 4:1 combination of oleic acid and erucic acid, with thrombocytopenia as the main side effect and sometimes leading to a hemorrhagic diathesis. We studied platelet count, size and membrane surface exposure of platelet activation antigens in 17 adult X-ALD patients. Eight patients used the prescribed amount of erucic acid (as glyceroltrierucate) or more (very compliant), five used less(compliant), and four did not use the diet. All eight very compliant patients had highly enlarged platelets and seven manifested thrombocytopenia. An enhanced in vivo platelet activation status was established by increased platelet surface expression of P-selectin (CD62P, PADGEM, GMP-140) in five of the seven thrombocytopenic patients, and of increased fibrinogen receptor exposure (measured with the antibody PAC-1) in three of these five patients. The other nine compliant or untreated patients had normal platelet counts and, generally, normal P-selection and fibrinogen receptor expression. A diet-induced 7- to 27-fold enrichment of erucic acid was observed in the platelets of the four patients studied. We conclude that the thrombocytopenia in AMN patients using Lorenzo'soil is associated with circulating platelets that have an increased erucic acid content, size and activation status. We hypothesize that the erucic acid in some way induces the increased size and thus, directly or indirectly, increased platelet activation or instability in vivo. This then causes the thrombocytopenia, with circulating platelets representing a population that has not yet been sufficiently changed to be removed, but has clear signs of activation.

Can flow cytometric detection of platelet activation early in pregnancy predict the occurrence of preeclampsia? A prospective study.

OBJECTIVES: An increased platelet activation status is present in patients with preeclampsia. Our purpose was (1) to establish by means of flow cytometry whether platelets circulate in an activated state during the first and second trimesters of pregnancy and (2) to establish whether early platelet activation predicts the onset of preeclampsia. STUDY DESIGN: Consecutively, 244 pregnant women were included in a prospective study design. Platelets in whole blood samples from the pregnant women in the first trimester, the second trimester, and after delivery were labeled with the following antibodies associated with platelet activation: anti-CD62P (P-selectin, alpha-granule secretion), anti-CD63 (GP53, lysosomal secretion), anti-CD31 (GPIIa', platelet endothelial cell adhesion molecule-1). The surface antigen exposure was determined by double-label flow cytometry with anti-CD42b (GPIb, a platelet-specific monoclonal glycoprotein) to select platelets and platelet-derived materials. Preeclampsia was defined as a diastolic blood pressure > or = 90 mm Hg and proteinuria > or = 0.3 gm in a 24-hour urine sample (International Society for Study of Hypertension in Pregnancy criteria). RESULTS: Seventeen of 244 patients had preeclampsia (6.9%). Only first-trimester CD63 expression had an area under the curve > 0.5 by receiver-operator characteristic curve analysis and was selected as a possible predictor of preeclampsia. We found a sensitivity of 47% and a specificity of 76% with use of a percentage of activated platelets above 2% as a positive test. Likelihood ratios were 1.94 for positive likelihood and 0.69 for negative likelihood. Univariate logistic regression analysis results were odds ratio 2.8 (95% confidence interval 1.0 to 7.6). Multivariate logistic regression analysis results were odds ratio 2.9 (95% confidence interval 0.92 to 8.9). However, the odds ratio of first antenatal diastolic blood pressure was two to four times higher than the odds ratio of first-trimester CD63 expression. The combination of first-trimester CD63 and first antenatal diastolic blood pressure increases the positive likelihood ratio from 1.94 to 9.4, with a sensitivity of 41%, a specificity of 96%, and a negative likelihood ratio of 0.62. CONCLUSIONS: Increased first-trimester CD63 expression is an independent risk factor for development of preeclampsia. CD63 expression might be useful to identify a subgroup of patients with a high risk for development of preeclampsia, especially in combination with first-trimester antenatal diastolic blood pressure. This method of patient selection may enable more efficient intervention studies in patients at risk than do the selection methods used so far.

Extensive platelet activation in preeclampsia compared with normal pregnancy: enhanced expression of cell adhesion molecules.

OBJECTIVES: Platelets play an important role in the pathophysiologic mechanisms of preeclampsia. Our purpose was to investigate by means of flow cytometry to what extent platelets circulate in an activated state during normal pregnancy and whether this activation is more extensive in preeclampsia. STUDY DESIGN: Platelets in whole blood from 10 preeclamptic third-trimester pregnant women (highest diastolic blood pressure range 100 to 130 mm Hg, proteinuria range 0.59 to 11.5 gm/24 hr) and from 10 normotensive third-trimester pregnant controls were analyzed with the following activation markers: anti-P-selectin (alpha-granule secretion), anti-CD63 (lysosomal secretion), PAC-1 (monoclonal antibody against fibrinogen receptor conformation of the glycoprotein IIb/IIIa complex), anti-platelet endothelial cell adhesion molecule-1, and annexin-V (a placental protein that binds to negatively charged phospholipids, present on the outside of the platelet plasma membrane after activation). The differences in surface antigen exposure between the two groups were determined by double-label flow cytometry. Flow cytometric data were analyzed in two ways: first, the percentages of activated platelets above a certain threshold compared with a nonpregnant control sample were determined, indicative for activation of a subpopulation of cells, and, second, the mean fluorescence intensities were determined, indicative of the mean surface antigen expression of the total platelet population. RESULTS: Analysis of the percentage of activated platelets proved most informative. With this analysis an enhanced platelet activation status was present in 4 of 10 normotensive patients and a more extensive platelet activation status in all 10 preeclamptic patients, as indicated by P-selectin (p = 0.008) and CD63 (p = 0.03) expression. Increased platelet endothelial cell adhesion molecule-1 (p = 0.005) expression was also observed in preeclampsia. CONCLUSIONS: Flow cytometric analysis clearly indicated that platelets circulate in a more extensively activated state during preeclampsia than during normal pregnancy. The increased platelet endothelial cell adhesion molecule-1 expression in preeclamptic patients demonstrates that, besides alpha-granular and lysosomal release, other hitherto unknown mechanisms are involved. Platelet endothelial cell adhesion molecule-1 appears to be the best marker to distinguish preeclamptic patients from normotensive pregnant women. Only a subpopulation of the platelets appears to be activated.