Age estimation with cemental incremental lines in normal and periodontally diseased teeth using phase contrast microscope: an original research

Background Age estimation is an important factor in forensic science for human identification. Teeth are considered to play a vital role as they resist decomposition at death unlike other tissues. This resistance and the gradual structural changes that take place throughout the life of an individual have made teeth useful indicators for age estimation. Dental cementum shows continuous apposition throughout the life of an individual. Tooth cemental annulation is a microscopic method for the determination of an individual’s age based on the analysis of incremental lines of cementum. Light microscopy as well as specialized microscopic methods have been employed to enhance the assessment of the cemental annulations. Periodontal disease is the most common dental problem affecting millions of people. Assessing the efficiency of the tooth cemental annulations method in periodontally diseased teeth is an important requisite. This study aims at assessing and evaluating the tooth cemental annulations in normal and periodontally diseased teeth using phase contrast microscopic method for age determination. Results A total of 60 teeth were included in the study and out of which 30 teeth were normal (sound teeth without any associated pathologies) and 30 were periodontally involved teeth respectively. Longitudinal ground sections were prepared and observed under phase contrast microscope. Measurements were made using an image analyzer software. The total width of the cementum was divided by the distance between two incremental lines. The eruption age of the tooth was then added to this to obtain the chronologic age for each individual. The results in the present study showed that tooth cemental annulations are applicable to periodontally sound teeth as well as in periodontally diseased teeth. There was no significant difference of estimated age from the actual age in both periodontally sound and periodontally diseased teeth. Normal teeth showed a reliability value of 92% and periodontally compromised teeth showed 96% respectively. There was no substantial influence of periodontal health on the estimated age. Conclusions The study concludes that the use of phase contrast microscopy in conjunction with image enhancement procedures improves the accuracy of age estimation and may serve as a reliable aid in forensic identification.

PSXV-13 The activity of thiol cathepsins in seminal plasma of stallions with different percentages of viable sperm after freezing-thawing

The aim of this work was to study the activity of cathepsins B, L and H in the seminal plasma of stallions with normal (>25%, n = 11) and low (< 25%, n = 13) percentage of viable sperm after freezing-thawing. Sperm of 24 Arabian stallions aged from 5 to 20 years (12.1 ± 4.8 years on average) was collected during the breeding season (February-May). The activity of cathepsins in spermoplasm was studied by the spectrofluorometric method (System 3 Scanning Spectrofluorometry, Optical technology devices, inc. Elmstord, New York, 10523) by Barrett&Kirschke. The viability of the sperm was determined after its freezing-thawing. Sperm smears were eosin stained and live:dead-ratio was examined using an Olympus BX41 phase contrast microscope (Olympus Corporation, Japan). The significance of differences in the studied groups was determined using the Mann-Whitney U-test. There were no significant differences in the activity of catepsins B and L in the spermoplasm of stallions with normal and low percentages of viable spermatozoa after freezing-thawing. It was found that in the group of stallions with a normal percentage of viable spermatozoa after freezing-thawing, the activity of cathepsin H in the spermoplasm was significantly higher than in the group with a low percentage of viable spermatozoa (P = 0.0219). Free radicals formed during freezing-thawing of sperm can damage cell membranes, leading to loss of sperm viability. Thiol cathepsins are involved in the degradation of oxidatively modified proteins and, apparently, it is cathepsin H that is most actively involved in this process in the sperm of stallions. We assume that the low activity of cathepsin H in the seminal plasma of stallions with a low percentage of viable spermatozoa indicates a high involvement of this enzyme in the protection of sperm membranes from oxidative damage. The research was supported by the Russian Science Foundation grant No. 20-16-00101.

Automated Confluence Measurement Method for Mesenchymal Stem Cell from Brightfield Microscopic Images

Cell confluence is an important metric in cell culture, as proper timing is essential to maintain cell phenotype and culture quality. To estimate cell confluence, transparent cells are observed under a phase-contrast or differential interference contrast microscope by a biologist, whose estimations are error-prone and subjective. To overcome the necessity of using the phase-contrast microscope and reducing intra- and inter-observer errors, we have proposed an algorithm that automatically measures cell confluence by using a commonly used brightfield microscope. The proposed method consists of a transport-of-intensity equation-based brightfield microscopic image processing, an image reconstruction method, and an adaptive image segmentation method based on edge detection, entropy filtering, and range filtering. Experimental results have shown that our method has outperformed several popular algorithms, with an F-score of 0.84 ± 0.07, in images with various cell confluence values. The proposed algorithm is robust and accurate enough to perform confluence measurement with various lighting conditions under a low-cost brightfield microscope, making it simple and cost-effective to use for a fully automated cell culture process.

Behavior of Cell Passing Through Micro Slit Between Micro Machined Plates

Deformation of each cell, as it passes through the micro-slit in the flow channel, has been investigated in vitro. A slit with a rectangular cross section (height 10 μm, width 0.4 mm, length 0.1 mm) was made in the center of the flow path by photolithography technique. Myoblasts (C2C12: mouse myoblast cell line) were used for the test. The flow rate of the medium, in which the cells were suspended, was controlled by a pressure head between the inlet and the outlet. Deformation of each cell passing through the micro-slit was observed with an inverted phase contrast microscope. Using the contour of the image of each cell passing through the slit intermittently, several parameters were analyzed: the two-dimensional projected area, the degree of deformation by ellipse approximation, and the deformation direction. The experimental results show that elongation of the cell in the slit tends to decrease the area of the cell.

Evaluation of the Toxicity on Lung Cells of By-Products Present in Naphthalene Secondary Organic Aerosols

In 2018, seven million people died prematurely due to exposure to pollution. Polycyclic aromatic hydrocarbons (PAHs) are a significant source of secondary organic aerosol (SOA) in urban areas. We investigated the toxic effects of by-products of naphthalene SOA on lung cells. These by-products were 1,4-naphthoquinone (1,4-NQ), 2-hydroxy-1,4-naphthoquinone (2-OH-NQ), phthalic acid (PA) and phthaldialdehyde (OPA). Two different assessment methodologies were used to monitor the toxic effects: real-time cell analysis (RTCA) and the Holomonitor, a quantitative phase contrast microscope. The chemicals were tested in concentrations of 12.5 to 100 µM for 1,4-NQ and 1 to 10 mM for 2-OH-NQ, PA and OPA. We found that 1,4-NQ is toxic to cells from 25 to 100 µM (EC50: 38.7 µM ± 5.2); 2-OH-NQ is toxic from 1 to 10mM (EC50: 5.3 mM ± 0.6); PA is toxic from 5 to 10 mM (EC50: 5.2 mM ± 0.3) and OPA is toxic from 2.5 to 10 mM (EC50: 4.2 mM ± 0.5). Only 1,4-NQ and OPA affected cell parameters (migration, motility, motility speed and optical volume). Furthermore, 1,4-NQ is the most toxic by-product of naphthalene, with an EC50 value that was one hundred times higher than those of the other compounds. RTCA and Holomonitor analysis showed a complementarity when studying the toxicity induced by chemicals.