Multispectral imaging flow cytometry with spatially and spectrally resolving snapshot-mosaic cameras for the characterization and classification of bioparticles

In the development and optimization of biotechnological cultivation processes the continuous monitoring through the acquisition and interpretation of spectral and morphological properties of bioparticles are challenging. There is therefore a need for the parallel acquisition and interpretation of spatially and spectrally resolved measurements with which particles can be characterized and classified in-flow with high throughput. Therefore, in this paper we investigated the scientific and technological connectivity of standard imaging flow cytometry (IFC) with filter-on-chip based spatially and spectrally resolving snapshot-mosaic cameras for photonic sensing and control in a smart and innovative microfluidic device. For the investigations presented here we used the microalgae Haematococcus pluvialis (HP). These microalgae are used commercially to produce the antioxidant keto-carotenoid astaxanthin. Therefore, HP is relevant to practically demonstrate the usability of the developed system for Multispectral Imaging Flow Cytometry (MIFC) platform. The extension of standard IFC with snapshot-mosaic cameras and multivariate data processing is an innovative approach for the in-flow characterization and derived classification of bioparticles. Finally, the multispectral data acquisition and the therefore developed methodology is generalizable and enables further applications far beyond the here characterized population of HP cells

The Future of Capital Punishment in Florida: Analysis and Recommendations

The Supreme Court\u27s decision abolishing the death penalty, at least as it existed in most jurisdictions, hardly represents the final resolution of the controversy over capital punishment. Given substantial public sentiment which apparently favors capital punishment in some form-voiced, for example, in the results of the recent referendum in California-various legislative bodies will face the question of whether capital punishment can and should be legislatively reinstated. In December 1972 the State of Florida became the first jurisdiction to pass judgment on this question. The legislature enacted a bill allowing imposition of the death penalty in certain circumstances. The two articles which follow highlight the competing policy and legal considerations which face legislatures after Furman, and illustrate one state\u27s response to those considerations. The first article is a slightly revised version of a memorandum to the Florida Governor\u27s Committee to Study Capital Punishment, submitted by the Committee\u27s Legal Advisory Staff. It sketches the various possible legislative responses to Furman and recommends one. The second indicates other recommendations made to the legislators and comments on the ultimate legislative determination

From microtiter plates to droplets - there and back again

Droplet-based microfluidic screening techniques can benefit from interfacing established microtiter plate-based screening and sample management workflows. Interfacing tools are required both for loading preconfigured microtiter-plate (MTP)-based sample collections into droplets and for dispensing the used droplets samples back into MTPs for subsequent storage or further processing. Here, we present a collection of Digital Microfluidic Pipetting Tips (DMPTs) with integrated facilities for droplet generation and manipulation together with a robotic system for its operation. This combination serves as a bidirectional sampling interface for sample transfer from wells into droplets (w2d) and vice versa droplets into wells (d2w). The DMPT were designed to fit into 96-deep-well MTPs and prepared from glass by means of microsystems technology. The aspirated samples are converted into the channel-confined droplets’ sequences separated by an immiscible carrier medium. To comply with the demands of dose-response assays, up to three additional assay compound solutions can be added to the sample droplets. To enable different procedural assay protocols, four different DMPT variants were made. In this way, droplet series with gradually changing composition can be generated for, e.g., 2D screening purposes. The developed DMPT and their common fluidic connector are described here. To handle the opposite transfer d2w, a robotic transfer system was set up and is described briefly

Airway remodeling: The Drosophila model permits a purely epithelial perspective

Airway remodeling is an umbrella term for structural changes in the conducting airways that occur in chronic inflammatory lung diseases such as asthma or chronic obstructive pulmonary disease (COPD). The pathobiology of remodeling involves multiple mesenchymal and lymphoid cell types and finally leads to a variety of hardly reversible changes such as hyperplasia of goblet cells, thickening of the reticular basement membrane, deposition of collagen, peribronchial fibrosis, angiogenesis and hyperplasia of bronchial smooth muscle cells. In order to develop solutions for prevention or innovative therapies, these complex processes must be understood in detail which requires their deconstruction into individual building blocks. In the present manuscript we therefore focus on the role of the airway epithelium and introduce Drosophila melanogaster as a model. The simple architecture of the flies’ airways as well as the lack of adaptive immunity allows to focus exclusively on the importance of the epithelium for the remodeling processes. We will review and discuss genetic and environmentally induced changes in epithelial structures and molecular responses and propose an integrated framework of research for the future

Male-female communication triggers calcium signatures during fertilization in Arabidopsis

Cell-cell communication and interaction is critical during fertilization and triggers free cytosolic calcium ([Ca2+](cyto)) as a key signal for egg activation and a polyspermy block in animal oocytes. Fertilization in flowering plants is more complex, involving interaction of a pollen tube with egg adjoining synergid cells, culminating in release of two sperm cells and their fusion with the egg and central cell, respectively. Here, we report the occurrence and role of [Ca2+](cyto) signals during the entire double fertilization process in Arabidopsis. [Ca2+](cyto) oscillations are initiated in synergid cells after physical contact with the pollen tube apex. In egg and central cells, a short [Ca2+](cyto) transient is associated with pollen tube burst and sperm cell arrival. A second extended [Ca2+](cyto) transient solely in the egg cell is correlated with successful fertilization. Thus, each female cell type involved in double fertilization displays a characteristic [Ca2+](cyto) signature differing by timing and behaviour from [Ca2+](cyto) waves reported in mammals

Male-female communication triggers calcium signatures during fertilization in Arabidopsis

Cell-cell communication and interaction is critical during fertilization and triggers free cytosolic calcium ([Ca2+](cyto)) as a key signal for egg activation and a polyspermy block in animal oocytes. Fertilization in flowering plants is more complex, involving interaction of a pollen tube with egg adjoining synergid cells, culminating in release of two sperm cells and their fusion with the egg and central cell, respectively. Here, we report the occurrence and role of [Ca2+](cyto) signals during the entire double fertilization process in Arabidopsis. [Ca2+](cyto) oscillations are initiated in synergid cells after physical contact with the pollen tube apex. In egg and central cells, a short [Ca2+](cyto) transient is associated with pollen tube burst and sperm cell arrival. A second extended [Ca2+](cyto) transient solely in the egg cell is correlated with successful fertilization. Thus, each female cell type involved in double fertilization displays a characteristic [Ca2+](cyto) signature differing by timing and behaviour from [Ca2+](cyto) waves reported in mammals

Versatile regularisation toolkit for iterative image reconstruction with proximal splitting algorithms

Ill-posed image recovery requires regularisation to ensure stability. The presented open-source regularisation toolkit consists of state-of-the-art variational algorithms which can be embedded in a plug-and-play fashion into the general framework of proximal splitting methods. The packaged regularisers aim to satisfy various prior expectations of the investigated objects, e.g., their structural characteristics, smooth or non-smooth surface morphology. The flexibility of the toolkit helps with the design of more advanced model-based iterative reconstruction methods for different imaging modalities while operating with simpler building blocks. The toolkit is written for CPU and GPU architectures and wrapped for Python/MATLAB. We demonstrate the functionality of the toolkit in application to Positron Emission Tomography (PET) and X-ray synchrotron computed tomography (CT)

Supervillin modulation of focal adhesions involving TRIP6/ZRP-1

Cell–substrate contacts, called focal adhesions (FAs), are dynamic in rapidly moving cells. We show that supervillin (SV)—a peripheral membrane protein that binds myosin II and F-actin in such cells—negatively regulates stress fibers, FAs, and cell–substrate adhesion. The major FA regulatory sequence within SV (SV342-571) binds to the LIM domains of two proteins in the zyxin family, thyroid receptor–interacting protein 6 (TRIP6) and lipoma-preferred partner (LPP), but not to zyxin itself. SV and TRIP6 colocalize within large FAs, where TRIP6 may help recruit SV. RNAi-mediated decreases in either protein increase cell adhesion to fibronectin. TRIP6 partially rescues SV effects on stress fibers and FAs, apparently by mislocating SV away from FAs. Thus, SV interactions with TRIP6 at FAs promote loss of FA structure and function. SV and TRIP6 binding partners suggest several specific mechanisms through which the SV–TRIP6 interaction may regulate FA maturation and/or disassembly