A new fractionator principle with varying sampling fractions: exemplified by estimation of synapse number using electron microscopy

The quantification of ultrastructure has been permanently improved by the application of new stereological principles. Both precision and efficiency have been enhanced. Here we report for the first time a fractionator method that can be applied at the electron microscopy level. This new design incorporates a varying sampling fraction paradigm. The method allows for systematic random sampling from blocks of variable slab thickness, thereby eliminating the need for exhaustive serial sectioning through an entire containing space. This novel approach acknowledges the inaccuracy inherent in estimating the total object number using section sampling fractions based on the average thickness of sections of variable thicknesses. As an alternative, this approach estimates the correct particle section sampling probability based on an estimator of the Horvitz–Thompson type, resulting in a theoretically more satisfying and accurate estimate of the expected number of particles for the defined containing space.     

Assessment of particle retention and clearance in the intrapulmonary conducting airways of hamster lungs with the fractionator1

A modified version of the fractionator was used to estimate the total number of polystyrene microspheres retained in the airways of hamster lungs at two different time points after inhalation. A systematic three-stage subsampling procedure with known sampling fractions was adopted. First, each lung was cut into slices, from which primary disectors were sampled systematically with a known sampling fraction. From each primary disector, smaller sub-disectors were subsampled, and the corresponding sampling fraction was estimated by point counting. Finally, a few particles were counted at the microscopic level in the sub-disectors, and the final estimate of total particle number (which is unbiased irrespective of any tissue deformations) was easily computed as a product of the counted number times the reciprocal of the successive sampling fractions. The error variance of each estimate was assessed from the data using a new estimator. An average of 6% of the deposited particles were retained on the epithelial surface of the intrapulmonary conducting airways shortly after the inhalation, from which at least one-third was already phagocytosed by macrophages. After 24 h, an average of 87% of the particles retained shortly after the inhalation had been cleared. The proportion of particles ingested by macrophages had increased to at least 87%, in three out of four animals studied.     

On the empirical variance of a fractionator estimate

The fractionator consists of several sampling stages with systematic sampling at each stage; data are collected only at the last stage. Therefore, predicting the error variance of a fractionator estimator is a non-trivial problem, because the observations are correlated in a complicated, unknown way. Gundersen proposed to split the material sampled at the first stage into two subsets, and to compute the variance of the pooled estimate empirically using the corresponding pair of observations made in these two subsets. The idea is very effective, but the estimator thus proposed needed some corrections. The purpose of this paper is to present an improved estimator of the coefficient of error of a fractionator estimator using Gundersen's design.     

AKT regulates IL-1β-induced proliferation and activation of hepatic stellate cells

Background: Activated hepatic stellate cells (HSCs) are closely involved in the initiation, perpetuation, andresolution of liver fibrosis. Pro-inflammatory cytokine levels are positively correlated with the transition from liverinjury to fibrogenesis and contribute to HSC pathophysiology in liver fibrosis. Methods: In this study, we investigatedthe effect of the pro-inflammatory cytokine interleukin (IL)-1β on the proliferation and signaling pathways involvedin fibrogenesis in LX-2 cells, an HSC cell line, using western blotting and cell proliferation assays. Results: IL-1βincreased the proliferation rate and α-smooth muscle actin (SMA) expression of LX-2 cells in a dose-dependentmanner. Within 1 h after IL-1β treatment, c-Jun N-terminal kinase (JNK), p38, and nuclear factor-κB (NF-κB)signaling was activated in LX-2 cells. Subsequently, protein kinase B (AKT) phosphorylation and an increase in α-SMA expression were observed in LX-2 cells. Each inhibitor of JNK, p38, or NF-κB decreased cell proliferation, AKTphosphorylation, and α-SMA expression in IL-1β-treated LX-2 cells. Conclusion: These results indicate that JNK,p38, and NF-κB signals converge at AKT phosphorylation, leading to LX-2 activation by IL-1β. Therefore, the AKTsignaling pathway can be used as a target for alleviating liver fibrosis by the inflammatory cytokine IL-1β.     

Estimating the total number of lymphatic valves in infant lungs with the fractionator

The fractionator is illustrated by means of a biomedical example involving the estimation of the number of lymphatic valves in lungs of infants who had died from sudden infant death syndrome (SIDS) and other known causes. The method is unbiased irrespective of tissue deformations and it does not require external information such as section thickness. An upper bound of the coefficient of error of the estimate of the number of valves within one lung was 6.5%, despite the fact that the number of valves counted per lung at the last stage ranged between 11 and 37 only. The upper bound includes the biological variation of the number of valves among infant lungs. Some theoretical remarks are also made on the efficiency of the fractionator. It is suggested, for instance, that the initial sampling stages cause more impact on the precision of the final estimator than the subsequent stages, and that an optimal arrangement of fragments submitted to systematic sampling should have the smallest fragments at the ends, with fragment contents increasing smoothly toward the middle of the series.     

Using biased image analysis for improving unbiased stereological number estimation – a pilot simulation study of the smooth fractionator

The smooth fractionator was introduced in 2002. The combination of a smoothing protocol with a computer‐aided stereology tool provides better precision and a lighter workload. This study uses simulation to compare fractionator sampling based on the smooth design, the commonly used systematic uniformly random sampling design and the ordinary simple random sampling design. The smooth protocol is performed using biased information from crude (but fully automatic) image analysis of the fields of view. The different design paradigms are compared using simulation in three different cell distributions with reference to sample size, noise and counting frame position. Regardless of clustering, sample size or noise, the fractionator based on a smooth design is more efficient than the fractionator based on a systematic uniform random design, which is more efficient than a fractionator based on simple random design. The fractionator based on a smooth design is up to four times more efficient than a simple random design.     

The changes of gap junctions between pituitary folliculo‐stellate cells during the postnatal development of zucker fatty and lean rats

We investigated the effect of leptin on the postnatal development of gap junctions between folliculo‐stellate cells by using Zucker fatty (fa/fa) rats that have defects of the functional leptin receptor. Male Zucker fatty rats (fa/fa) and male Zucker lean rats (+/+) were used at each of the following postnatal ages: 20, 30, 40, 50, 60, 70, 80, 90 days, and 1 year. On one of the aforementioned dates, the anterior pituitary glands were prepared for observation by transmission electron microscopy. We quantified the number of follicles and gap junctions, and calculated the rate of occurrence as the ratio of the number of gap junctions existing between folliculo‐stellate cells per intersected follicular profile. In Zucker lean male rats, the number of gap junctions remained relatively constant from days 50 to 90 (0.44 ± 0.02 to 0.49 ± 0.03), and was similar in 1 year old rats (0.47 ± 0.03). These data were statistically higher compared to Zucker fatty male rats. In Zucker fatty male rats, very few gap junctions were observed in 30‐day‐old rats (0.04 ± 0.01: mean ± SE). This disruption of gap junction formation persisted, and the number of gap junctions remained constant and showed a low level from days 40 to 90 (0.11 ± 0.02 to 0.17 ± 0.02); this finding was similar in 1‐year‐old rats (0.17 ± 0.02). These observations indicate that the effect of leptin over the gap junction formation within the anterior pituitary glands was directly mediated by interaction with the functional leptin receptor present on the folliculo‐stellate cells. Microsc. Res. Tech. 77:31–36, 2014. © 2013 Wiley Periodicals, Inc.     

Measuring microenvironment mechanical stress of rat liver during diethylnitrosamine induced hepatocarcinogenesis by atomic force microscope

We developed a highly sensitive method to detect liver tissue stiffness with atomic force microscopy (AFM), and investigated the physical features of hepatocarcinogenesis. Wistar rats received weekly intraperitoneal injections of diethylnitrosamine (DEN) or saline (control) followed by a 2-week wash-out period. Liver samples were harvested at 10, 14, or 18 weeks for pathological examination and stress detection. Previously normal liver tissues developed fibrosis and carcinoma after DEN administration. Although the elastic modulus (E) values of the normal (saline; 0.18 ± 0.04 MPa), fibrotic (8 weeks DEN; 0.25 ± 0.06 MPa) and cirrhotic (12 weeks DEN; 0.39 ± 0.06 MPa) tissues were significantly different, there was no significant difference between the E values of the cirrhotic and the hepatic cell carcinoma (16 weeks DEN; 0.42 ± 0.07 MPa) tissues. Thus, tissue stiffness quantitatively increases during hepatocarcinogenesis, and AFM can be used to sensitively and precisely detect liver stiffness at the microscopic level. Microsc. Res. Tech. 2009. © 2009 Wiley-Liss, Inc.     

Unbiased stereological estimation of the rat fetal pituitary volume and of the total number and volume of TSH cells after maternal dexamethasone application

Glucocorticoids have an inhibitory influence on proliferation activity of the pituitary cells while stimulating apoptosis. Therefore, it was hypothesized that the synthetic glucocorticoid, dexamethasone (DX), has an inhibitory influence on the number of thyroid‐stimulating hormone (TSH) cells during fetal development. The effects of maternal administration of DX on stereological parameters of TSH cells, and TSH serum concentration were investigated in 21‐day‐old rat fetuses. On day 16 of pregnancy, the experimental dams received 1.0 mg DX/kg b.w. subcutaneously, followed by 0.5 mg DX/kg b.w./day on days 17 and 18 of gestation. The control gravid females received the same volume of saline vehicle. TSH cells were stained immunocytochemically by the peroxidase–antiperoxidase (PAP) method. The fetal pituitary volumes were estimated using Cavalieri's principle. A physical disector counting technique in combination with the fractionator sampling method was used for estimation of pituitary TSH cell number. Cell and nuclear volumes were measured with a planar rotator. Maternal DX application was found to cause a significant decrease of pituitary volume and number of TSH cells per pituitary in 21‐day‐old fetuses in comparison with the control fetuses. TSH cell number expressed per body weight unit declined significantly after maternal DX administration. These results indicate an inhibitory DX influence on proliferative activity of precursors and likely differentiated TSH cells and increased apoptotic prevalence. The histological appearance, volume of TSH cells and TSH serum concentration suggest intensive synthetic activity in TSH cells of DX exposed fetuses. Microsc. Res. Tech. 73:1077–1085, 2010. © 2010 Wiley‐Liss, Inc.     

Unbiased and efficient estimation of the total number of terminal bronchiolar duct endings in lung: a modified physical disector

A novel modification of the physical disector is described which was used to estimate the total number of terminal bronchiolar duct endings (TBDEs) in human infant lung. TBDEs are closed three-dimensional space curves of complex shape that are inherently difficult to count from histological sections. However, careful consideration of the microanatomy of the terminal duct endings provides us with the opportunity to define a very simple and unbiased counting rule. To apply the rule in practice we also need to determine a suitable disector height. Owing to the complex shape of the TBDE we had no prior knowledge of what disector height would be suitable for counting the TBDE structures. Exhaustive serial sectioning of complete TBDE structures was carried out and showed that any disector height under 90 μm would give unbiased counts. A further empirical study was then undertaken to determine the most efficient disector height. This was found to be 50 μm.
The total number of TBDEs in the upper lobe of the right lung of six human infants aged between 13 and 25 weeks was also estimated. The estimates of numerical density obtained with our modification of the physical disector were multiplied by estimates of lung lobe volume obtained using Cavalieri's Principle. The total number of TBDEs in the lobes ranged from 15 323 to 57 768, with a mean of 40 306. The average coefficient of error of the number estimates was 19%, which was deemed precise enough given the biological coefficient of variation between TBDE number of 36%.     

Comparative study of DNA synthesis and nucleolar organizer regions of sinusoid littoral cells in mouse regenerating liver.

Variations in DNA synthesis (DNAs) and Nucleolar Organizer Regions (NORs) were studied in the littoral cell population from regenerating liver of C3HS inbred mice standardized for periodicity analysis. Immunohistochemical detection of Bromodeoxyuridine (BrdU) with a monoclonal antibody and silver staining of NORs (AgNORs) were assessed by means of a digital image analysis system in histological sections. Tissue samples were obtained every four hours from the 30th to the 54th hours after a partial hepatectomy. The results showed, in both parameters, a gradual increment of the values during the period studied, with highest values (DNAs 107.1 +/- 16.1 SE; AgNORs 77.3 +/- 3.4 SE) located at 16:00/54 Time of Day / Hours Post-Hepatectomy (TD/HPH), which were significantly different (p <0.001) from the values of the first sample (DNAs 38.1 +/- 9.5 SE; AgNORs 27.3 +/- 1.0 SE) taken at 16:00/30 TD/HPH. The results of our experiment demonstrate the existence of a strong correlation of DNA synthesis measured by BrdU immunohistochemistry and AgNORs numbers in sinusoid littoral cells from mouse regenerating liver.     

Use of multiphoton microscopy to diagnose liver cancer and lung metastasis in an orthotopic rat model

Liver or lung biopsy for suspicious lesions has several disadvantages such as bleeding, bile leak or pneumothorax, needle track seeding, and time-consuming histopathological procedure. The ability to directly observe cellular and subcellular details and then perform "optical biopsy" is a major goal in the development of new interventional techniques. Multiphoton microscopy (MPM) enables real-time noninvasive visualization of tissue architecture and cell morphology in live tissue. We performed a study to evaluate whether MPMcan make real-time optical diagnosis for liver cancer and lung metastasis using an orthotopic rat model with Morris hepatoma. We found that real-time high-resolution MPMimaging could clearly show tissue architecture and cell morphology. In the normal liver tissue, MPMimaging clearly revealed the blood-filled sinusoids and cords of hepatocytes. In the cancerous tissue, MPMimaging clearly illustrated that cancer cells displayed marked cellular and nuclear pleomorphism. MPMimages were comparable to golden standard hematoxylin-eosin staining images. Moreover, MPMimaging had deep penetration with the capability of optical sectioning. In short, MPMcan make real-time optical diagnosis for liver cancer and lung metastasis. This study provides the groundwork for further using multiphoton endoscopy to perform real-time noninvasive "optical biopsy" for liver cancer and lung metastasis in the near future.     

Estimation of the section thickness and optical disector height with a simple calibration method

The distance between the upper and lower surfaces of a section (i.e. the section thickness) can be measured with a microcator or a shaft encoder. In the present report, an alternative simple method is described for estimating the section thickness where such equipment is not available. The basic principle of the method is based on a calibration method already described in the literature. The main difference is that it enables one to make more precise measurements. Provided that the calibration and measurements are made properly, this method can be used in estimating the section thickness, optical disector heights, and in particular in the determination of the thickness sampling fraction for the optical fractionator.     

显微图像的复杂粘连细胞个数统计

显微细胞图像分析包括对生物医学图像进行识别测定,统计计数。文中对复杂的粘连严重细胞进行处理和个数统计。首先利用细胞的色度信息对图像二值化,用链码表示出轮廓。然后通过凹点分割略微粘连的细胞,再用面积法统计粘连严重细胞和单个细胞的个数。误差小于2%。结论表明对于粘连严重的细胞,该方法比传统的腐蚀算法,单纯的凹点分割有效。     

Elemental concentrations in air-exposed and vacuum-stored cryosections of rat liver cells

Elemental concentrations in different compartments of cryosections of isolated rat liver cells cryotransferred and freeze-dried were compared with those obtained after storage under vacuum for 12 or 60 h and after exposure to room air for 2 min. Poorer image contrast and segregation artefacts are frequently found in air-exposed sections, together with a slight but significant decrease of the K concentration in the cytoplasm and an increase of the S concentration in the liver cell nuclei and the extracellular medium. Extreme distortions of both ultrastructure and elemental distributions are observed if the sections are even slightly colder than the surrounding atmosphere. While storage of frozen-dried cryosections under vacuum for less than 12 h does not lead to alterations in the sections, gross changes are found both in morphology and elemental distribution in sections stored under vacuum for about 60 h. Long-time vacuum storage of frozen-dried cryosections is, therefore, not recommended.     

Estimation of loss probability and the admissible number of users in a network link considering a lognormal multifractal traffic model with exponential variance

In this work, we propose an analytical expression for estimating the byte loss probability at a single server queue with multifractal traffic arrivals. Initially, we address the theory concerning multifractal processes, especially the Hölder exponents of the multifractal traffic traces. Next we focus our attention on the second order statistics of multifractal traffic processes. More specifically, we assume that an exponential model is adequate for representing the variance of the traffic process under different time scale aggregation. Then, we compare the performance of the proposed approach to other relevant approaches. In addition, based on the results of the analysis, we propose a new admission control strategy that takes into account the multifractal traffic characteristics. We compare the proposed admission control strategy to some other widely used admission control methods. The simulation results show that the proposed loss probability estimation method is accurate, and the proposed admission control strategy is robust and efficient.     

Microspectroscopic measurement of the optical properties of rat liver in the visible region

Microspectroscopy is used to investigate optical properties of haemoglobin-free perfused rat liver. Visible spectra of 20 μm diameter spot size were measured in transmission and/or reflection modes as a function of the thickness (< 1200 μm) of the liver-edge. Optical density (OD) in transmission mode increased with the increasing liver thickness, whereas in reflection mode OD decreased but became almost constant above a certain thickness (c. 600 μm) of the liver. The Kubelka-Munk (KM) two-flux model, with a minor modification, was applied successfully to the analysis of the changes in OD as a function of the thickness. This approach estimates the KM absorption coefficient (E_KM), KM scattering coefficient (S_KM) and effective penetration depth (δ_eff) of the liver. The optical properties were similar to reported values, obtained with different methods.     

Accurate assessment of nonalcoholic fatty liver disease lesions in liver allograft biopsies by a smartphone platform: A proof of concept

Macrovesicular steatosis (MS) is a major risk factor for liver graft failure after transplantation and pathological microscopic examination of a frozen tissue section remains the gold standard for its assessment. However, the latter requires an experienced in‐house pathologist for correct and rapid diagnosis as well as specific equipment that is not always available. Smartphones, which are must‐have tools for everyone, are very suitable for incorporation into promising technology to generate moveable diagnostic tools as for telepathology. The study aims to compare the microscopic assessment of nonalcoholic fatty liver disease (NAFLD) spectrum in liver allograft biopsies by a smartphone microscopy platform (DIPLE device) to standard light microscopy. Forty‐two liver graft biopsies were evaluated in transmitted light, using an iPhone X and the microscopy platform. A significant correlation was reported between the two different approaches for graft MS assessment (Spearman's correlation coefficient: r = .93; p < .001) and for steatohepatitis feature (r = .56; p < .001; r = .45; p < .001). Based on these findings, a smartphone integrated with a cheap microscopy platform can achieve adequate accuracy in the assessment of NAFLD in liver graft and could be used as an alternative to standard light microscopy when the latter is unavailable.