Microscope Stage Research Articles

Gyöngysor-dramaturgia

This paper introduces the children’s theatre created in 1978 on the Microscope Stage under Péter Levente’s leadership and analyses the form and style of the children’s performances connected to the performance space. The ‘string of pearls’ dramaturgy developed here and refined by dramaturg Ildikó Döbrentey centred on ‘acting together’, on moderated coexistence. The subject of this paper is exploring this method.

Instantaneous tension measurements in droplet interface bilayers using an inexpensive, integrated pendant drop camera.

Recently, droplet interface bilayers (DIBs) have been used to determine bilayer tension and thickness in situ by automated image analysis using a microscope and an applied voltage. In this paper, we demonstrate improvements to these measurements by integrating an inexpensive pendant drop setup onto the microscope stage, which allows for simultaneous imaging of DIBs from both the bottom and side. By using pendant drop shape analysis in situ to determine the monolayer tension of the droplets, we avoid the reliance on applied voltages to determine tension. The integrated system also allows for direct measurement of both the major and minor diameter of the elliptical contact region, which produces a more direct measurement of the bilayer specific capacitance. Additionally, we demonstrate a technique for measuring the instantaneous monolayer tension of DIBs using shape analysis despite the assumed requirement for axial symmetry in pendant drop tensiometry. Compared to previous DIB measurements, the integrated pendant drop-microscope system provides improved accuracy accompanied by a fivefold to twentyfold improvement in precision while considerably decreasing the experiment time.

Sufficient condition for reliability measurement of sample geometry by atomic force microscopy

A sufficient condition for determining the reliability of geometry measurements using atomic force microscopy for relatively small cantilever tilt angles is proposed. A relationship between the basic geometric parameters of surface roughness, geometric deviations of the probe, the angles of the cantilever and the inclination of the side faces of the probe, as well as the dimensions of the nonlocal point of the probable contact of its side faces with protrusions of roughness has been established. As a sufficient condition for the reliability of geometry measurements using atomic force microscopy, an obvious requirement is accepted. It determines the smallness of the ratio of the sizes of a nonlocal point to the distance between neighboring nonlocal points. Publications in which the measurement of surface nano-geometry of the samples does not indicate the roughness of the sample surface and the probe, the angles at the tip of the probe and the tilt of the cantilever, as well as the best resolution (smallest step) at which the study is carried out, cannot be accepted as reliable, because the results obtained in them are probabilistic in nature. The surface images obtained using atomic force microscopy without proper justification for the resolution (value of the measurement step) represent only a qualitative picture, on the basis of which it makes no sense to carry out any computational manipulations. In order to increase the reliability of measurements of surface geometry using atomic force microscopy, it is necessary to radically increase the accuracy of the manufacture of probes, as well as use probes with the smallest possible angle at the apex. In addition, it is necessary to make changes in the design of the atomic force microscopy. In particular, the automatic rotation of the microscope stage should be designed. It should provide closeness the probe axis direction to the normal to the average plane of the sample. This “integral” angle of rotation of the microscope stage is easily iteratively determined at the stage of preliminary investigation of the geometry of the surface of the sample. In this case, it will be necessary to geometrically increase the length of the cantilever so that the base extends beyond the limits of the sample.

Acoustophoretic assembly of millimeter-scale Janus fibers.

This article presents a method for the assembly of millimeter-scale Janus fibers using acoustophoresis as an assembly mechanism. An acoustic flow cell mounted to a 3D printer combines acoustophoresis and additive manufacturing in a unique approach that allows for the assembly of textured Janus fibers. A dispersion consisting of polymethylmethacrylate (PMMA) filler particles in a UV curable polymer resin is passed through an acoustically excited capillary tube. To fundamentally understand this process, we develop a suspension balance model that accounts for acoustophoresis and concentration-driven shear-induced diffusion. Once assembled, the particle-polymer dispersion is cured using UV illumination to create a polymer composite fiber with particles immobilized on one side in a Janus-like configuration. The Janus fiber is observed to modify the light transmission profile when rotated on an optical microscope stage. Tensile measurements of the fiber show that the Young's modulus of the Janus fiber (50.5 MPa) is approximately twice that of a fiber fabricated from the polymer alone (24.7 MPa). The process we describe here could serve as a pathway for the fabrication of a variety of functional Janus fibers with possible applications to wearable textiles, soft robotics or surgical sutures.

Whole slide imaging system using deep learning-based automated focusing.

The auto focusing system, which involves moving a microscope stage along a vertical axis to find an optimal focus position, is the chief component of an automated digital microscope. Current automated focusing algorithms, especially those deployed in cost effective microscopy systems, often cannot match the efficiency of a skilled human operator in keeping a sample in focus. This work presents an auto focusing system that utilises the recent advances in machine learning, namely deep convolutional neural networks (CNN). It improves upon prior work in this domain. The results of the focusing algorithm are demonstrated on an open data set. We describe the practical implementation of this method on a low cost digital microscope to create a whole slide imaging system (WSI). Results of a clinical study using this WSI system are presented. The study demonstrates the efficacy of this system in a practical scenario.

Photosynthetic activity in two heteromorphic life‐history stages of a freshwater red alga, Thorea gaudichaudii (Thoreales) from Japan, in response to an irradiance and temperature gradient

SUMMARYWe determined the effect of irradiance and temperature on the photosynthesis of two heteromorphic life‐history stages of an endangered freshwater red alga, Thorea gaudichaudii (Thoreales) by laboratory and field measurements. Net oxygenic photosynthesis–irradiance models of macroscopic and microscopic life‐history stages revealed similar low irradiance‐adapted responses, with a compensation irradiance (Ec) of 6.71 and 2.56 μmol photons m−2 s−1 (4.30–9.13 and 0.13–7.19, 95% Bayesian prediction interval, BPI) and saturating irradiance (Ek) of 26.6 and 30.0 μmol photons m−2 s−1 (19.0–37.4 and 12.1–63.0, BPI), respectively. A temperature‐dependent model of net photosynthesis and dark respiration in macroscopic and microscopic stages also showed similar temperature responses, and the gross photosynthetic rate (GPmax), 3.54 and 6.34 μg O2 gww−1 min−1 (3.10–3.99 and 5.31–8.21, BPI), was highest at 32.1 and 35.7°C (29.8–34.0 and 29.5–48.6, BPI). The maximum quantum yields (F v/F m) in macroscopic and microscopic stages were also similar in response with respect to temperature; however, it was somewhat steady at low temperatures with the highest value of 0.54 and 0.62 (0.54–0.55 and 0.61–0.63, BPI) at 17.8 and 15.0°C (16.7–18.8 and 12.3–17.1, BPI). The effective quantum yield (Φ PSII) in macroscopic and microscopic stages was also negatively correlated with irradiance, which decreased after 12 h of continuous exposure to 50 (low) and 1000 (high) μmol photons m−2 s−1 at 12 and 22°C. Large declines of Φ PSII and subsequent failure of F v/F m recovery were particularly enhanced at high irradiance, signifying photoinhibition. Diurnal change of Φ PSII and incident irradiance of the macroscopic stage under the field measurement revealed the midday depression of Φ PSII; however, there was little direct sunlight due to shading by the trees, and algae were occurring in the shaded locations in the freshwater spring.

Impact of Lymph Node Sampling in Stage II and III Epithelial Ovarian Cancer Patients with Clinically Negative Lymph Nodes

The main surgical treatment of stage II and III epithelial ovarian cancer (EOC) is complete cytoreduction, while the main role of lymph node sampling is to exclude microscopic stage III disease in an apparent stage I EOC. This study aims to evaluate the impact of lymph node sampling in stage II and III EOC patients with clinically negative lymph nodes. This is a retrospective cohort study including 51 stage II and III EOC patients treated and followed up between 2012 and 2016. They were treated by complete cytoreduction. Sixteen cases had lymph node sampling, while it was not done in 35 cases. The study was performed at National Cancer Institute (NCI)-Cairo University. There was no statistically significant difference regarding overall survival (P value 0.649) or disease-free survival (P value 0.372) between the group of patients who had lymph node sampling and the other group of patients who had no lymph node sampling. Lymph node ratio (LNR) was not associated with a statistically significant impact regarding overall survival or disease-free survival. There is no impact of lymph node sampling on stage II and III EOC patients with clinically negative lymph nodes.

A Software Tool for High-Throughput Real-Time Measurement of Intensity-Based Ratio-Metric FRET

Förster resonance energy transfer (FRET) is increasingly used for non-invasive measurement of fluorescently tagged molecules in live cells. In this study, we have developed a freely available software tool MultiFRET, which, together with the use of a motorised microscope stage, allows multiple single cells to be studied in one experiment. MultiFRET is a Java plugin for Micro-Manager software, which provides real-time calculations of ratio-metric signals during acquisition and can simultaneously record from multiple cells in the same experiment. It can also make other custom-determined live calculations that can be easily exported to Excel at the end of the experiment. It is flexible and can work with multiple spectral acquisition channels. We validated this software by comparing the output of MultiFRET to that of a previously established and well-documented method for live ratio-metric FRET experiments and found no significant difference between the data produced with the use of the new MultiFRET and other methods. In this validation, we used several cAMP FRET sensors and cell models: i) isolated adult cardiomyocytes from transgenic mice expressing the cytosolic epac1-camps and targeted pmEpac1 and Epac1-PLN sensors, ii) isolated neonatal mouse cardiomyocytes transfected with the AKAP79-CUTie sensor, and iii) human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) transfected with the Epac-SH74 sensor. The MultiFRET plugin is an open source freely available package that can be used in a wide area of live cell imaging when live ratio-metric calculations are required.

Mélanome au stade ganglionnaire : analyse du ganglion sentinelle, curage ganglionnaire, perspective des traitements adjuvants. Enquête nationale française sur les pratiques actuelles et envisagées

The recent publication of randomized trials investigating the efficacy of adjuvant therapy and completion lymph node dissection at microscopic stage III melanoma calls for a reappraisal of melanoma management from different angles: indications for sentinel lymph node biopsy, indications for completion lymph node dissection in microscopic-stage disease, and adjuvant therapies. Our objective was to evaluate current practices and to question French onco-dermatologists about any changes they envisaged in their practices in the light of recent publications. We conducted a national survey among members of the Cutaneous Oncology Group of the French Society of Dermatology in October 2017. Forty French health centers were included, and 53individual responses were collected. Sentinel lymph node biopsy for melanoma was performed at 75% of the centers. Before the summer of 2017and the publication of MSLT-II (proving the absence of any therapeutic benefits for complete lymph node dissection in microscopic stage III melanoma), when a positive sentinel lymph node was diagnosed, immediate completion lymph node dissection was performed at 90% of the centers. After the publication of MSLT-II, 45% of the respondents considered stopping this practice. The risk-benefit ratio prompted prescription of nivolumab and of combined dabrafenib+trametinib as adjuvant therapy by respectively 96% and 79% of respondents, while the corresponding rates for interferon and ipilimumab were only 21% and 15%. Early melanoma management stands on the verge of major changes thanks to the arrival of efficient adjuvant therapies and a decrease in immediate completion lymph node dissections for patients with microscopic stage III is also anticipated.

Characterization and Correction of the Geometric Errors using a Confocal Microscope for Extended Topography Measurement, Part II: Experimental Study and Uncertainty Evaluation

This paper presents the experimental implementations of the mathematical models and algorithms developed in Part I. Two experiments are carried out. The first experiment determines the correction coefficients of the mathematical model. The dot grid target is measured, and the measurement data are processed by our developed and validated algorithms introduced in Part I. The values of the coefficients are indicated and analyzed. Uncertainties are evaluated using the Monte Carlo method. The second experiment measures a different area of the dot grid target. The measurement results are corrected according to the coefficients determined in the first experiment. The mean residual between the measured points and their corresponding certified values reduced 29.6% after the correction. The sum of squared errors reduced 47.7%. The methods and the algorithms for raw data processing, such as data partition, fittings of dots’ centers, K-means clustering, etc., are the same for the two experiments. The experimental results demonstrate that our method for the correction of the errors produced by the movement of the lateral stage of a confocal microscope is meaningful and practicable.

Phase Dependent Vectorial Current Control in Symmetric Noisy Optical Ratchets.

In this work, we demonstrate single microparticle transport in a symmetric noisy optical ratchet made with a linear array of 20 optical potentials, where each potential is a spatially symmetric low power (<2.5 mW) three-dimensional trap. Both the external force F(t) and the depth V_{0i}(t) of the optical potentials are dynamic and change at the same frequency ν=2 Hz. The depths of the individual optical potentials are random (uncorrelated noise) distributed around a mean value V_{0}, ⟨V_{0i}(t)⟩=V_{0}, while the external force is periodic and unbiased ⟨F(t)⟩=0. The system is completely symmetric for times t≫1/ν. Directed transport is possible as a result of the symmetry being broken at times on the order of 1/ν. We find that the direction and speed of motion (current) are coupled to the phase difference between the noise in the optical potentials and the external periodic force.

The correlation between coal char structure and reactivity at rapid heating condition in TGA and heating stage microscope

The coal pyrolysis and char gasification process have been widely studied in TGA at a low heating rate, which is far lower than the heating rate when coal particle enters into the boiler and gasifier. In this work, the isothermal CO2 gasification reactivity of in-situ coal chars was performed in rapid heating TGA and a heating stage microscope. The char structure parameters determined by XRD and Raman were combined to describe the char reactivity at the same temperature. Results showed that maximum weight loss rate (DTGmax) of raw coal evidently increased and the corresponding maximum rate temperature raised with the increasing of heating rate during pyrolysis. Nonetheless, the heating rate had slightly effect on gasification reactivity of in-situ char samples when it exceeded 50 K/min. Considering the physical meaning and universality of the coal char structure and gasification reactivity fitting, the XRD and Raman structural parameters were combined to describe the gasification reactivity. The new method for coal char reactivity under rapid heating rate is established, Rs=0.174+43.14d002,aLc,a+3.58ID3×ID4IG2. Furthermore, a visually heating stage microscope was also applied for determining the in-situ char reactivity through image analyzing. The gasification process of in-situ coal char particles fits the shrinking particle pattern at the original and midterm stages. However, the shrinking particle pattern changed into the shrinking core pattern at high carbon conversion. Moreover, the CO inhibition effect and inter-particle interaction account for the difference of gasification reactivity by rapid heating TGA and heating stage microscope.

Interfacial microrheology and tensiometry in a miniature, 3-d printed Langmuir trough

We introduce a new approach for manipulating and studying complex fluid interfacial systems, that enables simultaneous tensiometry to measure surface pressure and high-performance interfacial shear microrheology. In contrast to existing techniques, we use a miniature Langmuir trough produced by 3-d printing methods, which fits on the stage of a conventional inverted microscope and which allows compression and stretching of the fluid-fluid interfaces at a constant bulk fluid volume. The tensiometry measurement relies on the quantitative imaging of a curved meniscus separate from the flat interface being imaged under the microscope. We first demonstrate that this approach can measure the interfacial tension of simple fluid-fluid and polymer-laden interfaces with an accuracy comparable to the pendant drop apparatus and Wilhelmy plates. We then study the mechanics of an adsorbed protein layer formed at an air-water (a-w) interface by simultaneously measuring its surface pressure and its interfacial viscosity. To perform two-point interfacial microrheology, micrometer-sized probes spread on the interface are tracked to 5 nm precision with very low drift, yielding measurements of interfacial viscosity having a wide dynamic range and high sensitivity (10-9-10-1Nsm-1).

Understanding fusibility characteristics and flow properties of the biomass and biomass-coal ash samples

The fusibility characteristics and flow properties of the biomass and biomass-coal ash samples were investigated in this paper. Ash fusibility of the biomass-coal blended samples was evaluated systematically. The mineral matters variation and liquidus temperature of the biomass-coal blended samples were demonstrated by FactSage. The results show that the ash fusion temperature of biomass-coal blended samples decreases and then increases with the increasing content of biomass (straw). The ash fusion temperature reaches the minimum when straw content is 50%. Moreover, the liquidus temperature reflected in the phase diagram can predict the variation of ash fusion temperature well. Furthermore, the flow properties of high temperature ash (HTA) and low temperature ash (LTA) straw samples at different atmosphere were observed using the heating stage microscope. In the heating process, the HTA and LTA straw samples show the similar variation: shrinkage, fusion and spreading. However, the volume changes of the HTA and LTA straw samples are different considerably attributing to the mineral matters variation. It was revealed that the volume change of HTA and LTA straw samples is not obvious at the lower temperature. For the LTA straw sample in air atmosphere, the maximal volume change is obtained.

Ice recrystallisation and melting in ice cream with different proteins levels and subjected to thermal fluctuation

Ice cream of high quality has smooth consistency due to ice crystal sizes between 20 and 50 μm, which depends on ice cream formulation and thermal stress during storage. Ice crystals of 3 “traditional” (milk proteins and stabilisers) and 2 vegan (inulin and potato proteins) ice cream formulations were studied using a cold stage microscope under frozen conditions. Samples were observed before and after a temperature fluctuation (cycles of −18 °C and −6 °C for 14 days), and the data obtained compared with the results of a melting test. Results showed that milk proteins and stabilisers conferred structural stability to ice cream, both in terms of small crystal size (20–50 μm) and behaviour during melting, suitable for storage. Ice creams without one of the two constituents or in presence of potato proteins or inulin, showed a crystal size increase; in this case ice creams were for immediate consumption.

The urogenital papillae as an indicator of sexual maturity in male African catfish, Clarias gariepinus, (Burchell, 1822)

The present study investigated a non-invasive method based on macroscopic morphological features of male Clarias gariepinus for the assessment of sexual maturity stage. As African catfish cannot be stripped for semen as in most other fish species; they have to be killed to obtain semen from the testes. This method could be used to ensure that males used to obtain sperm are sexually mature to increase the concentration and quality of mature sperm for egg fertilization in the production of C. gariepinus. Morphological features including the length and width of the urogenital papillae (UGP), an external secondary sexual characteristic of C. gariepinus (n = 89) were measured and calculated as a percentage of total body length. The urogenital papillae length (LUGP) and width (WUGP) index of each fish were used and compared with the macroscopic and microscopic maturity stage of the testes. The results revealed that although positive, a significant correlation does not exist between the LUGP or WUGP and maturity stage of the testes. Therefore, although this procedure would be valuable as an initial non-invasive assessment of maturity, it does not provide an accurate or reliable indication of the maturity stage of the fish.

Characterization of Lauric Acid Precipitated from Biocompatible Solvents

Water resistance of lauric acid (L) dissolved in biocompatible solvents mainly depended on the water affinity of solvent. L in DMSO (DL) was most sensitive to water and higher than L in N-methyl pyrrolidone (NL) and L in 2-pyrolidone (PL), respectively. From scanning electron microscope, differential scanning calorimetry, thermogravimetric analysis, powder x-ray diffractometry and hot stage microscope tests revealed the alteration of L crystal owing to the interference by solvent during precipitation. All L precipitates had lower melting point and degradation temperature than intact L in which L precipitated from 2-pyrrolidone exhibited the lowest melting temperature. These characteristics will be useful for modifying L in phase inversion in situ forming gel.

Fabrication of Microscope Stage for Vertical Observation with Temperature Control Function.

Samples are usually placed onto a horizontal microscope stage for microscopic observation. However, to observe the influence of gravity on a sample or study afloat behavior, it is necessary to make the microscope stage vertical. To accomplish this, a sideways inverted microscope tilted by 90° has been devised. To observe samples with this microscope, sample containers such as Petri dishes or glass slides must be secured to the stage vertically. A device that can secure sample containers in place on a vertical microscope stage has been developed and is described here. Attachment of this device to the stage allows observation of sample dynamics in the vertical plane. The ability to regulate temperature using a silicone rubber heater also permits observation of temperature-dependent sample behaviors. Furthermore, the temperature data is transferred to an internet server. Temperature settings and log monitoring can be controlled remotely from a PC or smartphone.

Fabrication of Microscope Stage for Vertical Observation with Temperature Control Function

Fabrication of Microscope Stage for Vertical Observation with Temperature Control Function

Novel pendrin inhibitor attenuates airway hyperresponsiveness and mucin expression in experimental murine asthma

Novel pendrin inhibitor attenuates airway hyperresponsiveness and mucin expression in experimental murine asthma