In-line Microscopy Research Articles

Membrane crystallization for recovery of lithium carbonate crystals: Study on process parameters and salts effect for Li2CO3-NaCl-KCl-LiCl solutions

In this work, membrane crystallization (MCr) process was employed for recovery of lithium carbonate (Li2CO3) crystals from synthetic brine solutions. First, the effect of the main operating conditions in MCr, including feed temperature (40, 50, and 60 °C) and flowrate (0.81, 1, and 1.3 L/min) was investigated on the crystallization of Li2CO3 in a binary solution. Next, the effect of main inorganic salts in the brine solutions (i.e., NaCl, KCl, and LiCl) and the mixture of them on the MCr performance and crystallization of Li2CO3 were investigated. In-line microscope, light microscope, scanning electron microscope, and X-ray diffraction test were used to observe the crystallization and characterize the obtained crystals. The obtained results revealed that feed temperature is the main parameter to dominate the nucleation and crystal formation and growth. Thus, the higher the feed temperature, the faster the crystal formation and the larger the crystals. The average crystal size increased from 9.59 to 30.51 μm, when the feed temperature increased from 40 to 60 °C, respectively. However, smaller crystals were observed with adding NaCl and KCl to the solution. Moreover, none of the operating parameters nor the salts additives did affect the crystals shape and the obtained Li2CO3 crystals possess needle-like shape.

Flow-through microscopy and image analysis for crystallization processes

The particle size and particle size distribution are crucial parameters for the final product application of active pharmaceutical compounds. Image analysis with inline microscopy is an important tool for monitoring and controlling the crystallization process. This work proposes a flexible and reliable image analysis algorithm that works by combining edge detection with intensity thresholding and implementing an advanced watershed. The code is made open source and should be easily implementable. Additionally, a simple flow-through image acquisition system is developed by modifying conventional optical microscopes. Finally, measurements of the particle size, the number concentration and the solid concentration are evaluated over a wide range of solid concentrations. These can be used to measure the nucleation rate and the yield. This research should reduce the barriers towards automated image analysis and provide new methods to monitor crystallization processes.

In meso in situ serial X-ray crystallography of soluble and membrane proteins at cryogenic temperatures.

Here, a method for presenting crystals of soluble and membrane proteins growing in the lipid cubic or sponge phase for in situ diffraction data collection at cryogenic temperatures is introduced. The method dispenses with the need for the technically demanding and inefficient crystal-harvesting step that is an integral part of the lipid cubic phase or in meso method of growing crystals. Crystals are dispersed in a bolus of mesophase sandwiched between thin plastic windows. The bolus contains tens to hundreds of crystals, visible with an in-line microscope at macromolecular crystallography synchrotron beamlines and suitably disposed for conventional or serial crystallographic data collection. Wells containing the crystal-laden boluses are removed individually from hermetically sealed glass plates in which crystallization occurs, affixed to pins on goniometer bases and excess precipitant is removed from around the mesophase. The wells are snap-cooled in liquid nitrogen, stored and shipped in Dewars, and manually or robotically mounted on a goniometer in a cryostream for diffraction data collection at 100 K, as is performed routinely with standard, loop-harvested crystals. The method is a variant on the recently introduced in meso in situ serial crystallography (IMISX) method that enables crystallographic measurements at cryogenic temperatures where crystal lifetimes are enormously enhanced whilst reducing protein consumption dramatically. The new approach has been used to generate high-resolution crystal structures of a G-protein-coupled receptor, α-helical and β-barrel transporters and an enzyme as model integral membrane proteins. Insulin and lysozyme were used as test soluble proteins. The quality of the data that can be generated by this method was attested to by performing sulfur and bromine SAD phasing with two of the test proteins.

Compact beam conditioning unit

A new modular beam conditioning unit is presented. The unit can be freely arranged to meet the requirements of the specific experimental setup. Currently the following modules are available: High precision beam position monitor, slit screens, filter wheel, ultra-fast shutter, beam stop and an in-line microscope. Special emphasis was put on the flexible and space saving design.

Optimized coherence parameters for high-resolution holographic microscopy

A holographic in-line microscope setup with a glass sample carrier commonly uses a coherent laser light source, with the disadvantage of an incomplete suppression of disturbing interferences and coherence-induced noise. We use temporally partially coherent and spatially partially coherent illumination in the micrometer range of 5 to 100 μm generated by spectrally and spatially filtered light-emitting diodes (LEDs) and tungsten filament lamps. It is shown how errors in reconstructed holograms are decreased by a factor of more than two and the spatial resolution reaches the limit as determined by the numerical aperture. A straightforward model is developed to investigate the effects of partial coherence. The optimization of the coherence parameters improves the imaging quality up to the limit of a conventional microscope.

Effect of zeta potential value on bacterial behavior during electrophoretic separation

The aggregation and/or adhesion of bacterial cells is a serious disadvantage of electrophoretic separations. In this study, physicochemical surface characteristics of bacteria were measured to establish their role in bacterial adhesion and aggregation on the basis of electrophoretic behavior of different clinical strains of Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli bacteria. The number and the shape of peaks obtained on the electropherograms were connected with the zeta potential measurements and in-line microscope observation using specially designed CE fluorescence stereomicroscope setup. These results suggest that the lower the zeta potential, the higher the number of smaller peaks detected. The direct microscopic observation of electrophoretic movement proved the presence of many small aggregates originating from individual or clustered bacterial cells. On the other hand, lower zeta potential was also observed for dead bacterial cells, which suggested that some of the peaks can be attributed to viable cells while the other to the dead ones.

Infrared lensless holographic microscope with a vidicon camera for inspection of metallic evaporations on silicon wafers

The lensless digital in-line microscope is proposed as a simple tool for post-production inspection of metallic film patterns on silicon wafers. Transmission holographic imaging is achieved by using a superluminescent diode emitting in the near infrared coupled into a single-mode high numerical aperture optical fiber and an infrared vidicon camera. Images of 300 nm thick vapor-deposited aluminum bolometers are presented.

Lensless digital holographic microscope with light-emitting diode illumination.

We demonstrate the operation of a digital in-line microscope with LED illumination. We show with a practical example that, for typical setups, the limited temporal coherence and the spatial incoherence of the source do not affect the resolving power of the system. On the contrary, important advantages are obtained in terms of signal-to-noise ratio and alignment simplification.

Filtration of lager beer with microsieves: flux, permeate haze and in-line microscope observations

Membrane fouling during filtration of lager beer with microsieves was studied through in-line microscope observations. It was observed that the main fouling was caused by micrometre-sized particles, presumably aggregated proteins. These particles formed flocks covering parts of the membrane surface. Most of the flocks could be removed by a strong temporary increase in crossflow. Underneath the flocks a permanent fouling layer was formed inside the pores. This made frequent removal of the flocks crucial in delaying the process of permanent in-pore fouling. Besides the fouling process the influence of pore size on permeate flux and turbidity was investigated. Centrifuged beer appeared to give a significantly clearer permeate than rough beer. For centrifuged beer and a microsieve with a pore diameter of 0.55 μm a haze of 0.23 EBC was obtained during 10.5 h of filtration at an average flux of 2.21×10 3 l/m 2 h. For a sieve with slit-shaped perforations of 0.70 μm×3.0 μm a haze of 0.46 EBC was obtained during 9 h of filtration at an average flux of 1.43×10 4 l/m 2 h. This flux is more than two-orders of magnitude higher than is commonly obtained with membrane-filtration of lager beer. Concentration of the beer by a factor of 12 hardly influenced the magnitude of the flux.

Depolarized fluorescence photobleaching recovery

The effects of the fact that the laser sources typically used in fluorescence photobleaching recovery (FPR) experiments in the most commonly employed in-line microscope imaging geometries, are highly linearly polarized, are examined in some detail. The implications of the results, in particular for the interpretation of FPR data in complex cell membrane systems in terms of laterally mobile and immobile sub-populations of the labelled molecular species of concern, are discussed. Methods of experimentally eliminating the potentially major rotational diffusion-based “artifacts”, different from those appropriate to three-dimensional (solution or suspension) systems which require other than in-line geometries, are delineated.