Comparison of different calibration methods for Mueller matrix microscopy of cells.

Mueller matrix (MM) imaging has demonstrated its potential application in much research, especially in probing delicate and complex biomedical specimens. Qualities of MM images are important for further quantitative characterization. In this paper, we compare the performance and imaging qualities of three calibration methods. Air, waveplate and cell specimen are selected as standard samples for comparison. In addition, we also propose two general MM imaging quality indices that can be used as quantitative evaluations for MM imaging systems and calculation processes based on real samples. The numerical calibration method turns out to give the best accuracy and precision, as well as the best image qualities.

Evaluating fibre orientation dispersion in white matter: Comparison of diffusion MRI, histology and polarized light imaging.

Diffusion MRI is an exquisitely sensitive probe of tissue microstructure, and is currently the only non-invasive measure of the brain's fibre architecture. As this technique becomes more sophisticated and microstructurally informative, there is increasing value in comparing diffusion MRI with microscopic imaging in the same tissue samples. This study compared estimates of fibre orientation dispersion in white matter derived from diffusion MRI to reference measures of dispersion obtained from polarized light imaging and histology. Three post-mortem brain specimens were scanned with diffusion MRI and analyzed with a two-compartment dispersion model. The specimens were then sectioned for microscopy, including polarized light imaging estimates of fibre orientation and histological quantitative estimates of myelin and astrocytes. Dispersion estimates were correlated on region - and voxel-wise levels in the corpus callosum, the centrum semiovale and the corticospinal tract. The region-wise analysis yielded correlation coefficients of r = 0.79 for the diffusion MRI and histology comparison, while r = 0.60 was reported for the comparison with polarized light imaging. In the corpus callosum, we observed a pattern of higher dispersion at the midline compared to its lateral aspects. This pattern was present in all modalities and the dispersion profiles from microscopy and diffusion MRI were highly correlated. The astrocytes appeared to have minor contribution to dispersion observed with diffusion MRI. These results demonstrate that fibre orientation dispersion estimates from diffusion MRI represents the tissue architecture well. Dispersion models might be improved by more faithfully incorporating an informed mapping based on microscopy data.

Computerized histomorphometric study of the splenic collagen polymorphism: A control-tissue for polarization microscopy.

Previous articles have pointed out the presence of type III collagen within the extracellular structure of the parenchymatous organs. This study aimed to quantitatively characterize the collagen polymorphism at the capsule and parenchymal trabeculae of the largest lymphoid organ of the body i.e., the spleen, in mouse, rat, and rabbit models. Following a Picrosirius Red-Polarization procedure and computer assisted image analysis of paraffin sections, the results showed (1) a predominant and significantly higher amount of type III collagen in the trabeculae area compared to the capsule area in the three species, (2) no statistical difference among the three species concerning the parenchymal collagen polymorphism or the type I/type III collagen ratio, (3) a heterogeneous type I/type III collagen ratio varying from 0.86 (mouse) to 6.62 (rabbit) in the fibromuscular capsule region. A qualitative analysis corroborated these histomorphometric results. In conclusion, the spleen may be used as (1) a control tissue to qualitatively visualize type I and III collagen under polarization microscopy and to validate the quality of PSR staining (2) an aid to accurately calibrate the angle of polarization before quantitative measurements of type I and type III collagen. Among the studied species, the rabbit spleen appeared to be the most appropriate control tissue as it showed the highest amount of type I collagen in the capsule and a similarly high amount of type III collagen in the parenchymal trabeculae.

Polarized Light Colposcopy Compared With Standard Colposcopy.

OBJECTIVE: The objective of this study was to determine the potential benefits of polarized light colposcopy compared with standard colposcopy examinations in the evaluation of women with abnormal cervical cytology. MATERIALS AND METHODS: Polarized and standard colposcopy examinations were performed on 330 subjects. Respective images and biopsy annotations were obtained. Sensitivity and specificity; differences in the severity of cervical neoplasia; agreement of colposcopy impression, biopsy intent, and biopsy site; and differences in the number of biopsies were determined using the ROC, Bowker's test of symmetry, kappa statistic, and paired t test, respectively. RESULTS: The sensitivity and specificity for a lesion being seen with nonpolarized light and polarized light colposcopy were 96.8% and 64.5%, and 96.8% and 64.9%, respectively. There was no statistically significant difference in the ROC of the lesion being seen between nonpolarized (80.7) and polarized (80.9) colposcopy. Likewise, there was no statistically significant difference in the ROC of intent to biopsy between nonpolarized (80.2) and polarized colposcopy (78.8). The agreement of cervical histopathology and colposcopy impression for nonpolarized and polarized colposcopy were 0.986 and 0.952, respectively. There was no significant difference between nonpolarized and polarized colposcopy in the mean number of lesions seen or number of sites intended to biopsy. CONCLUSIONS: Polarized light colposcopy was not useful as an adjunct to conventional colposcopy in this study. Further research needs to be performed to determine the overall utility of polarized light colposcopy in clinical practice.

Prevalence of giardiasis in children attending semi-urban daycare centres in Guatemala and comparison of 3 giardia detection tests.

Giardia intestinalis is an intestinal parasite widely prevalent in children attending daycare centres worldwide and has been associated with undernutrition. Stool samples from 48 Guatemalan children (aged 1.5-7 years) attending participating daycare centres were analyzed over five weeks for presence of Giardia intestinalis using light microscopy, ELISA, and rapid dipstick test. Giardia prevalence rates were 43.7% at Week 0 and 44.7% at Week 4, based on ELISA. Intensity, but not prevalence, of infection showed a trend toward decreased weight-for-age (1-tailed p = 0.08). We believe that ELISA analysis of stool samples may be further adapted for measuring the intensity of infection in humans.

Effects of buflomedil and pentoxifylline on hamster skin-flap microcirculation: prediction of flap viability using orthogonal polarization spectral imaging.

OBJECTIVE: This study investigated the effects of buflomedil and pentoxifylline, both of which are used in reconstructive surgery of hamster skin flap microcirculation, and evaluated the skin flap survival rate by orthogonal polarization spectral imaging. METHOD: Twenty-four adult male Syrian golden hamsters were divided into three groups: a control (C, 0.1 ml 0.9% saline), buflomedil (B, 3 mg/kg/day), and pentoxifylline group (P, 14.5 mg/kg/day). Treatments administered intraperitoneally were initiated 1 hour before skin flap preparation and continued for 7 days post-operatively at 12-hour intervals. Preparations (skin flaps) were divided into 12 fields, which were organized into six bands. Functional capillary density (FCD, in mm/mm(2)), distance from the skin flap base to blood flow cessation (Dist(with flow), in cm), percentage of viable skin (VA, in%), and qualitative analysis of blood flow by orthogonal polarization spectral imaging were performed at 1 and 24 hours and on the seventh post-operative day. RESULT: Bands IV, V, and VI presented no flow independent of time. The functional capillary density group B was higher than that of groups C and P, primarily after 24 hours. All groups showed an increase in D with time but reached similar final distances (C = 2.73, B = 2.78 and P = 2.70 cm). Moreover, the percentage of viable areas remained at approximately 50%. The orthogonal polarization spectral imaging was useful to assess viability by counting fields with and without blood flow. CONCLUSIONS: Functional capillary density values were higher in the buflomedil group compared to the control and pentoxifylline groups in this model. Functional capillary density did not influence D or the percentage of VA, and the technique showed favorable potential to assess/predict the viability of skin flaps within 1 h after surgery.

Effects of buflomedil and pentoxifylline on hamster skin-flap microcirculation: prediction of flap viability using orthogonal polarization spectral imaging

OBJECTIVE: This study investigated the effects of buflomedil and pentoxifylline, both of which are used in reconstructive surgery of hamster skin flap microcirculation, and evaluated the skin flap survival rate by orthogonal polarization spectral imaging. METHOD: Twenty-four adult male Syrian golden hamsters were divided into three groups: a control (C, 0.1 ml 0.9 percent saline), buflomedil (B, 3 mg/kg/day), and pentoxifylline group (P, 14.5 mg/kg/day). Treatments administered intraperitoneally were initiated 1 hour before skin flap preparation and continued for 7 days post-operatively at 12-hour intervals. Preparations (skin flaps) were divided into 12 fields, which were organized into six bands. Functional capillary density (FCD, in mm/mm²), distance from the skin flap base to blood flow cessation (Dist with flow, in cm), percentage of viable skin (VA, in percent), and qualitative analysis of blood flow by orthogonal polarization spectral imaging were performed at 1 and 24 hours and on the seventh post-operative day. RESULT: Bands IV, V, and VI presented no flow independent of time. The functional capillary density group B was higher than that of groups C and P, primarily after 24 hours. All groups showed an increase in D with time but reached similar final distances (C = 2.73, B = 2.78 and P = 2.70 cm). Moreover, the percentage of viable areas remained at approximately 50 percent. The orthogonal polarization spectral imaging was useful to assess viability by counting fields with and without blood flow. CONCLUSIONS: Functional capillary density values were higher in the buflomedil group compared to the control and pentoxifylline groups in this model. Functional capillary density did not influence D or the percentage of VA, and the technique showed favorable potential to assess/predict the viability of skin flaps within 1 h after surgery.

Fluorescence polarization standard for near infrared spectroscopy and microscopy.

We present studies of polarized absorption [linear dichroism (LD)] and fluorescence polarization of the styryl derivative (LDS 798) embedded in oriented poly(vinyl alcohol) (PVA) films. These films were oriented by progressive stretching up to eight folds. Both vertical and horizontal components of absorptions and fluorescence were measured and dichroic ratios were determined for different film stretching ratios. The dichroic ratio and fluorescence anisotropy values were analyzed as a function of PVA film stretching ratio by fitting according to the previously developed theory. For maximum stretching ratios, exceptionally high anisotropy (approximately 0.8) and polarization (approximately 0.9) values have been measured. The stretched films have high polarization values also for isotropic excitation in a wide spectral range (500-700 nm). Such films can be conveniently used as high polarization standards and we envision they will also have applications in near infrared (NIR) imaging microscopy, where they can be used for correcting an instrumental factor in polarization measurements.

Analysis of the analytical performance of laboratories participating in two major U.S. bulk asbestos proficiency testing programs.

Quality assurance and proficiency testing programs have been available for 27 years for U.S. laboratories using polarized light microscopy to analyze bulk building materials for asbestos. Total enrollment in the two principal bulk asbestos proficiency testing programs conducted by RTI International peaked at nearly 900 laboratories in the mid-1990s. Enrollment has stabilized in the last 5 years at approximately 475 laboratories, and rates of return of analysis results currently exceed 95%. More than 115,000 test samples have been sent worldwide by RTI to laboratories in these two programs. A review of more than 109,000 analysis results was undertaken to determine the frequencies of various qualitative error types, the types of bulk building materials most responsible for those errors, and the accuracy of precision of semiquantitative results. This assessment revealed that a small number of bulk building materials cause the majority of difficulties in each program, although error frequencies vary between the two programs. Overall laboratory performance has improved since the programs first began in the late 1980s, with a significant decline in qualitative errors and a much less dramatic decrease in errors associated with semiquantitative estimation of the amount of asbestos present.

Noninvasive in vivo assessment of the skeletal muscle and small intestine serous surface microcirculation in rat: sidestream dark-field (SDF) imaging.

The pathophysiology of microcirculation is intensively investigated to understand disease development at the microscopic level. Orthogonal polarization spectral (OPS) imaging and its successor sidestream dark-field (SDF) imaging are relatively new noninvasive optical techniques allowing direct visualization of microcirculation in both clinical and experimental studies. The goal of this experimental study was to describe basic microcirculatory parameters of skeletal muscle and ileal serous surface microcirculation in the rat using SDF imaging and to standardize the technical aspects of the protocol. Interindividual variability in functional capillary density (FCD) and small vessels (<25 microm in diameter) proportion was determined in anesthetized rats on the surface of quadriceps femoris (m. rectus femoris and m. vastus medialis) and serous surface of ileum. Special custom made flexible arm was used to fix the SDF probe minimizing the pressure movement artifacts. Clear high contrast images were analyzed off-line. The mean FCD obtained from the surface of skeletal muscle and ileal serous surface was 219 (213-225 cm/cm(2)) and 290 (282-298 cm/cm(2)) respectively. There was no statistically significant difference between rats in mean values of FCD obtained from the muscle (P = 0.273) in contrast to ileal serous surface, where such difference was statistically significant (P = 0.036). No statistically significant differences in small vessels percentage was detected on either the muscle surface (P = 0.739) or on ileal serous surface (P = 0.659). Our study has shown that interindividual variability of basic microcirculatory parameters in rat skeletal muscle and ileum is acceptable when using SDF imaging technique according to a highly standardized protocol and with appropriate fixation device. SDF imaging represents promising technology for experimental and clinical studies.

Over-diagnosis of malaria is not a lost cause.

BACKGROUND: Recent studies have highlighted the over-diagnosis of malaria in clinical settings in Africa. This study assessed the impact of a training programme implemented as part of an intervention trial on diagnostic behaviour of clinicians in a rural district hospital in a low-moderate malaria transmission setting. METHODS: From the beginning of 2005, a randomized controlled trial (RCT) of intermittent preventive treatment for malaria in infants (IPTi) has been conducted at the study hospital. As part of the RCT, the study team offered laboratory quality assurance, and supervision and training of paediatric ward staff using information on malaria epidemiology in the community. Data on clinical and blood slide confirmed cases of malaria from 2001 to 2005 were extracted from the hospital records. RESULTS: The proportion of blood slides positive for malaria parasites had decreased from 21% in 2001 to 7% in 2005 (p < .01). The proportion of outpatient and inpatient cases diagnosed as malaria ranged between 34% and 28% from 2001 to 2004 and this decreased substantially to 17% after the introduction of the package of training and support in 2005 (p < .01). There was no clear trend in the ratio of blood slide examined versus total diagnosis of malaria. CONCLUSION: It may be possible to change the diagnostic behaviour of clinicians by rigorous training using local malaria epidemiology data and supportive supervision.

Reader technique as a source of variability in determining malaria parasite density by microscopy.

BACKGROUND: Accurate identification and quantification of malaria parasites are critical for measuring clinical trial outcomes. Positive and negative diagnosis is usually sufficient for the assessment of therapeutic outcome, but vaccine or prophylactic drug trials require measuring density of infection as a primary endpoint. Microscopy is the most established and widely-used technique for quantifying parasite densities in the blood. METHODS: Results obtained by 24-27 expert malaria microscopists, who had independently read 895 slides from 35 donors, were analysed to understand how reader technique contributes to discrepancy in measurements of parasite density over a wide range of densities. RESULTS: Among these 35 donations, standard deviations ranged from 30% to 250% of the mean parasite density and the percent discrepancy was inversely correlated with the mean parasite density. The number of white blood cells indexed and whether parasites were counted in the thick film or thin film were shown to significantly contribute to discrepancy amongst microscopists. CONCLUSION: Errors in microscopy measurements are not widely appreciated or addressed but have serious consequences for efficacy trials, including possibly abandoning promising vaccine candidates.

Circularly polarized light standards for investigations of collagen fiber orientation in bone.

Bone exhibits positive form birefringence dominated by and dependent upon the orientation of its collagen. The biomechanical efficacy of bone as a tissue is largely determined by collagen fibers of preferred orientation and distribution (and corresponding orientation of mineral crystallites), and evidence is accumulating to demonstrate that this efficacy extends to function at the organ level. This study has three aims. The first is to provide a Background to the study of circularly polarized light (CPL) investigations of collagen fiber orientation in bone. The significance of preferred collagen fiber orientation in bone, linearly polarized light and CPL imaging principles, and a short history of CPL studies of mammalian functional histology are reviewed. The second is to describe, in some detail, methodological considerations relating to specimen preparation and imaging appropriate for the quantitative analysis of preferentially oriented collagen. These include section transparency, section thickness, the uniformity of the illuminating system, and CPL paraphernalia. Finally, we describe a grey-level standard useful for quantitative CPL, based upon mineralized turkey tendon, which shall be provided to investigators upon request. When due consideration is paid to specimen preparation and imaging conditions, quantitative assessment of collagen fiber orientation provides insight into the effects of mechanical loading on the skeleton.

Specificity and sensitivity of glaucoma detection in the Japanese population using scanning laser polarimetry.

AIMS: To investigate the usefulness of the scanning laser polarimeter (GDx; GDx Nerve Fiber Analyzer) for glaucoma detection in the Japanese population, and to investigate the difference in the thickness of retinal nerve fibre layer (RNFL) between normal tension glaucoma (NTG) and primary open angle glaucoma (POAG). METHODS: 69 eyes of 69 normal subjects and 115 eyes of 115 chronic open angle glaucoma patients (60 NTG and 55 POAG patients) were studied. The thickness of RNFL was measured with GDx. An eye was diagnosed as glaucomatous, if at least one original GDx variable showed p <5%. The difference in thickness of RNFL between the NTG and POAG groups was then investigated. RESULTS: 46 normal eyes (66.7%) were diagnosed as not glaucomatous (no variables showing p <5%), and 93 glaucomatous eyes (46 NTG and 47 POAG eyes) (80.9%) were diagnosed as glaucomatous. Actual values of average thickness, ellipse average, superior average, and superior integral were significantly lower in the POAG group than those in the NTG group. CONCLUSIONS: New variables which elucidate focal RNFL defects or early changes are needed to improve the moderate detection ability found in this present study. The pattern of the change in RNFL may differ in NTG and POAG groups.

The Cytoscan model E-II in intraoperative parathyroid gland identification in a rabbit model.

BACKGROUND: Intraoperative parathyroid gland identification and preservation is often a challenge even in the hands of experienced surgeons as they could be indistinguishable from fat or thyroid tissue. OBJECTIVE: The goal of this study was to demonstrate the use of the Cytoscan Model E-II, which uses orthogonal polarization spectral (OPS) imaging technology, as an intravital microscope in identifying parathyroid glands intraoperatively and differentiating the parathyroid glands from fat and thyroid tissue in a rabbit model. METHODS: The necks of 4 New England white rabbits were explored with the animals under a general anesthesia. The Cytoscan was used to obtain images of the vasculature of tissue suspected to be parathyroid, fat, and thyroid tissue. These were confirmed by histologic evaluation. RESULTS: All tissues were correctly identified by the Cytoscan and confirmed by histologic analysis. There was an obvious difference in the images obtained of fatty tissue as compared with parathyroid tissues. There was also an appreciable difference between parathyroid and thyroid tissue based on the difference in vascularity. CONCLUSIONS: OPS imaging technology can be used in identifying parathyroid glands based on the difference in vascularity from fat and the pattern and density of vessels when compared with thyroid tissue in a rabbit model. SIGNIFICANCE: The Cytoscan may play a future role in real time intraoperative identification of human parathyroid glands. Future investigation is warranted.

Spatially modulated illumination microscopy: online visualization of intensity distribution and prediction of nanometer precision of axial distance measurements by computer simulations.

During the last years, measurements considerably beyond the conventional "Abbe-Limit" of optical resolution in far field light microscopy were realized by several light microscopical approaches. Point spread function (PSF) engineering, spectral precision distance microscopy (SPDM), and related methods were used to demonstrate the feasibility of such measurements. SPDM allows the measurement of position and multiple distances between point-like fluorescent objects of different spectral signatures far below the optical resolution criterion as defined by the full width at half maximum of the PSF. Here, we report a software method to obtain online visualization of light distribution in the lateral and axial direction of any object detected in a spatially modulated illumination (SMI) microscope. This strongly facilitates routine application of SMI microscopy. The software was developed using Microsoft Visual C++ running on Windows NT. Furthermore, some aspects of the theoretical limits of the SPDM method were studied by virtual microscopy. For the case of SMI microscopy the precision of axial distance measurements was studied, taking into account photon statistics and image analysis procedures. The results indicate that even under low fluorescence intensity conditions typical for biological structure research, precise distance measurements in the nanometer range can be determined, and that axial distances in the order of 40 nm are detectable with such precision.

Quantitative in situ correlation between microscopic MRI and polarized light microscopy studies of articular cartilage.

OBJECTIVE: To establish the correlation between the non-invasive imaging by magnetic resonance microscopy (microMRI) and the histological imaging by polarized light microscopy (PLM) accurately, quantitatively, at the highest possible MRI resolution (13.7 microm), and based on the same piece of tissue (articular cartilage from canine shoulder joint). DESIGN: In microMRI experiments, the laminar appearance (the magic angle effect) of the proton intensity images and the anisotropic characteristics of the T(2)relaxation images were analysed. In PLM experiments, the images of the optical retardation and collagen-fibre orientation in cartilage were constructed in two dimensions. RESULTS: The T(2)profile has a distinctly asymmetric bell-shaped curve and three featured zones. The retardation profile has a non-zero minimum at the middle of the transitional zone of the tissue. The angle profile has a smooth variation across the transitional zone. These facts suggest that the collagen fibres in the transitional zone are not entirely random but have a residual order. In addition, the peak of the T(2)profile coincides with the minimum of the retardation profile, both represent the most isotropic region of the tissue. A hyperbolic tangent function was found to best describe the transition of the collagen fibres in cartilage. A set of criteria was developed for each technique to define the features in the quantitative measurements. CONCLUSIONS: The criteria offer, for the first time, a set of quantitative and objective means to subdivide the tissue thickness into the zones in histology and in MRI. It is shown that the microMRI zones based on the T(2)characteristics are statistically equivalent to the histological zones based on the collagen fibre orientation (t-probabilities of 0.730, 0.973, 0.647, 0.850 for the superficial, transitional, radial zones and the total thickness).

[Ensuring quality in the analyses of urinary calculi by a comparison of methods. 3d International Ring Trial]. / Sicherung der Qualität von Harnsteinanalysen durch Methodenvergleiche. 3. Internationaler Ringversuch.

Of 5 analysis tests of the 3rd International Ring Experiment for the control of the quality of methods of the analysis of urinary calculi 38 findings compiled according to 9 different techniques came in from 12 countries. The average deviation per component concerning all 5 analysis tests is considerably low with 0.10 mol proportions. The average quality measure SQ concerning all participants is approximately 2.00; it deteriorates from the X-ray diffraction method over the IR-technique to the other quantitative methods used. Advantages and disadvantages of the X-ray diffraction and IR-spectroscopic analysis are discussed. A methodically homogeneous and centralized performed analysis of urinary calculi shows advantages in the quality of the analyses.