Design and Construction of a Chamber Enabling the Observation of Living Cells in the Field of a Constant Magnetic Force

The aim of the work was to design and construct a microscopic stage that enables the observation of biological cells in a magnetic field with a constant magnetic force. Regarding the requirements for biological observations in the magnetic field, construction was based on the standard automatic stage of an optical microscope ZEISS Axio Observer, and the main challenge was to design a set of magnets which were the source of a field in which the magnetic force was constant in the observation zone. Another challenge was to design a magnet arrangement producing a weak magnetic field to manipulate the cells without harming them. The Halbach array of magnets was constructed using permanent cubic neodymium magnets mounted on a 3D printed polymer ring. Four sets of magnets were used, differing in their dimensions, namely, 20, 15, 12, and 10 mm. The polymer rings were designed to resist magnetic forces and to keep their shape undisturbed when working under biological conditions. To check the usability of the constructs, experiments with magnetic microparticles were executed. Magnetic microparticles were placed under the microscope and their movement was observed to find the acting magnetic force.

A Parallel Perifusion Slide From Glass for the Functional and Morphological Analysis of Pancreatic Islets

An islet-on-chip system in the form of a completely transparent microscope slide optically accessible from both sides was developed. It is made from laser-structured borosilicate glass and enables the parallel perifusion of five microchannels, each containing one islet precisely immobilized in a pyramidal well. The islets can be in inserted via separate loading windows above each pyramidal well. This design enables a gentle, fast and targeted insertion of the islets and a reliable retention in the well while at the same time permitting a sufficiently fast exchange of the media. In addition to the measurement of the hormone content in the fractionated efflux, parallel live cell imaging of the islet is possible. By programmable movement of the microscopic stage imaging of five wells can be performed. The current chip design ensures sufficient time resolution to characterize typical parameters of stimulus-secretion coupling. This was demonstrated by measuring the reaction of the islets to stimulation by glucose and potassium depolarization. After the perifusion experiment islets can be removed for further analysis. The live-dead assay of the removed islets confirmed that the process of insertion and removal was not detrimental to islet structure and viability. In conclusion, the present islet-on-chip design permits the practical implementation of parallel perifusion experiments on a single and easy to load glass slide. For each immobilized islet the correlation between secretion, signal transduction and morphology is possible. The slide concept allows the scale-up to even higher degrees of parallelization.

Automated Long-Term Monitoring of Parallel Microfluidic Operations Applying a Machine Vision-Assisted Positioning Method

As microfluidics has been applied extensively in many cell and biochemical applications, monitoring the related processes is an important requirement. In this work, we design and fabricate a high-throughput microfluidic device which contains 32 microchambers to perform automated parallel microfluidic operations and monitoring on an automated stage of a microscope. Images are captured at multiple spots on the device during the operations for monitoring samples in microchambers in parallel; yet the device positions may vary at different time points throughout operations as the device moves back and forth on a motorized microscopic stage. Here, we report an image-based positioning strategy to realign the chamber position before every recording of microscopic image. We fabricate alignment marks at defined locations next to the chambers in the microfluidic device as reference positions. We also develop image processing algorithms to recognize the chamber positions in real-time, followed by realigning the chambers to their preset positions in the captured images. We perform experiments to validate and characterize the device functionality and the automated realignment operation. Together, this microfluidic realignment strategy can be a platform technology to achieve precise positioning of multiple chambers for general microfluidic applications requiring long-term parallel monitoring of cell and biochemical activities.

Assessment of Cytological Atypia, AgNOR and Nuclear Area in Epithelial Cells of Normal Oral Mucosa Exposed to Toombak and Smoking

The purpose of this study was to assess cellular proliferative activity of clinically healthy oral mucosal epithelial cells of toombak dippers and smokers by means of AgNOR counts and nuclear areas via nuclear morphometry. Smears were collected from normal-appearing mouth floor mucosa and tongue of 75 toombak dippers, 75 smokers and 50 non-tobacco users between the ages of 20 and 70 with a mean age of 36 years. AgNORs were counted in the first 50 well-fixed, nucleated squamous cells and nuclear areas were calculated via microscopic stage micrometer. Cytological atypia was ascertained in 6 tobacco users and could not be ascertained in non-tobacco users. Statistically mean AgNOR numbers per nucleus in the non-tobacco users (2.45±0.30) was lower than the toombak dippers (3.081±0.39, p<0.004), and the smokers (2.715±0.39, p<0.02), and mean nuclear areas of epithelial cells of toombak dippers (6.081±0.39, p<0.009) and smokers (5.68±10.08, p<0.01) was also significantly higher than non-smokers (5.39±9.4). The mean number of nuclei having more than 3 AgNORs was 28%, 19% and 7% in toombak dippers, smokers and non-tobacco users, respectively. These findings support the view that toombak dipping and smoking are severe risk factors for oral mucosal proliferative lesions and exfoliative cytology is valid for screening of oral mucosal lesions.