BatchDeconvolution : a Fiji plugin for increasing deconvolution workflow

Deconvolution microscopy is a very useful, software-based technique allowing to deblur microscopy images and increase both lateral and axial resolutions. It can be used along with many of fluorescence microscopy imaging techniques. By increasing axial resolution, it also enables three-dimensional imaging using a basic wide-field fluorescence microscope. Unfortunately, commercially available deconvolution software is expensive, while freely available programs have limited capabilities of a batch file processing. In this work we present BatchDeconvolution, a Fiji plugin that bridges two programs that we used subsequently in an image deconvolution pipeline: PSF Generator and DeconvolutionLab2, both from Biomedical Imaging Group, EPFL. Our software provides a simple way to perform a batch processing of multiple microscopy files with minimal working time required from the user

Precise mass determination of single cell with cantilever-based microbiosensor system

Having determined the mass of a single cell of brewer yeast Saccharomyces cerevisiae by means of a microcantilever-based biosensor Cantisens CSR-801 (Concentris, Basel, Switzerland), it was found that its dry mass is 47,65 $\pm$ 1,05 pg. Found to be crucial in this mass determination was the cell position along the length of the cantilever. Moreover, calculations including cells positions on the cantilever provide a threefold better degree of accuracy than those which assume uniform mass distribution. We have also examined the influence of storage time on the single cell mass. Our results show that after 6 months there is an increase in the average mass of a single yeast cell

The influence of microinjection parameters on cell survival and procedure efficiency

Microinjection is a method commonly used to deliver various substances into cells. The procedure is performed on a widefield microscope stage using fine glass needle to penetrate the cell membrane. Microinjection can be carried out using a manual or semi-automatic mode. For commercially available equipment currently reported microinjection success rate and cell viability are relatively low (around 50% for both indicators). Here, for the first time, we systematically show how the microinjection effectiveness and cell viability are influenced by needle diameter and chosen microinjection mode. We found that manual mode entailed a higher injection rate, reducing cell viability at the same time. The reduction in needle diameter caused a significant increase in cell survival rate (from 43 to 73% for manual mode and from 58% to 86% for semi-automatic mode) and did not affect significantly the success rate. Our findings will help optimize this method in the context of cell biology research

Increasing AFM colloidal probe accuracy by optical tweezers

A precise determination of the cantilever spring constant is the critical point of all colloidal probe experiments. Existing methods are based on approximations considering only cantilever geometry and do not take into account properties of any object or substance attached to the cantilever. Neglecting the influence of the colloidal sphere on the cantilever characteristics introduces significant uncertainty in a spring constant determination and affects all further considerations. In this work we propose a new method of spring constant calibration for ‘colloidal probe’ type cantilevers based on the direct measurement of force constant. The Optical Tweezers based calibration method will help to increase the accuracy and repeatability of the AFM colloidal probe experiments

Vimentin association with nuclear grooves in normal MEF 3T3 cells

Vimentin, an intermediate filament protein present in leukocytes, blood vessel endothelial cells, and multiple mesenchymal cells, such as mouse embryonic fibroblasts (MEF 3T3), is crucial for various cellular processes, as well as for maintaining the integrity and durability (stability) of the cell cytoskeleton. Vimentin intermediate filaments (VIFs) adhere tightly to the nucleus and spread to the lamellipodium and tail of the cell, serving as a connector between the nucleus, and the cell’s edges, especially in terms of transferring mechanical signals throughout the cell. How these signals are transmitted exactly remains under investigation. In the presented work, we propose that vimentin is involved in that transition by influencing the shape of the nucleus through the formation of nuclear blebs and grooves, as demonstrated by microscopic observations of healthy MEF (3T3) cells. Grooved, or “coffee beans” nuclei, have, to date, been noticed in several healthy cells; however, these structures are especially frequent in cancer cells—they serve as a significant marker for recognition of multiple cancers. We observed 288 MEF3T3 cells cultured on polyhydroxyoctanoate (PHO), polylactide (PLA), and glass, and we identified grooves, coaligned with vimentin fibers in the nuclei of 47% of cells cultured on PHO, 50% of cells on glass, and 59% of cells growing on PLA. We also observed nuclear blebs and associated their occurrence with the type of substrate used for cell culture. We propose that the higher rate of blebs in the nuclei of cells, cultured on PLA, is related to the microenvironmental features of the substrate, pH in particular

Insights into in vitro wound closure on two biopolyesters-polylactide and polyhydroxyoctanoate

Two bio-based polymers have been compared in this study, namely: polylactide (PLA) and polyhydroxyoctanoate (PHO). Due to their properties such as biocompatibility, and biointegrity they are considered to be valuable materials for medical purposes, i.e., creating scaffolds or wound dressings. Presented biopolymers were investigated for their impact on cellular migration strategies of mouse embryonic fibroblasts (MEF) 3T3 cell line. Advanced microscopic techniques, including confocal microscopy and immunofluorescent protocols, enabled the thorough analysis of the cell shape and migration. Application of wound healing assay combined with dedicated software allowed us to perform quantitative analysis of wound closure dynamics. The outcome of the experiments demonstrated that the wound closure dynamics for PLA differs from PHO. Single fibroblasts grown on PLA moved 1.5-fold faster, than those migrating on the PHO surface. However, when a layer of cells was considered, the wound closure was by 4.1 h faster for PHO material. The accomplished work confirms the potential of PLA and PHO as excellent candidates for medical applications, due to their properties that propagate cell migration, vitality, and proliferation—essential cell processes in the healing of damaged tissue

Chromophore-assisted laser inactivation in cell biology

Chromophore-assisted laser inactivation (CALI) is a technique whereby engineered proteins and dye molecules that produce substantial amounts of reactive oxygen species upon absorption of light are used to perturb biological systems in a spatially and temporally defined manner. CALI is an important complement to conventional genetic and pharmacological manipulations. In this review, we examine the applications of CALI to cell biology and discuss the underlying photochemical mechanisms that mediate this powerful technique

Multiple paxillin binding sites regulate FAK function

Abstract Background FAK localization to focal adhesions is essential for its activation and function. Localization of FAK is mediated through the C-terminal focal adhesion targeting (FAT) domain. Recent structural analyses have revealed two paxillin-binding sites in the FAT domain of FAK. To define the role of paxillin binding to each site on FAK, point mutations have been engineered to specifically disrupt paxillin binding to each docking site on the FAT domain of FAK individually or in combination. Results These mutants have been characterized and reveal an important role for paxillin binding in FAK subcellular localization and signaling. One paxillin-binding site (comprised of α-helices 1 and 4 of the FAT domain) plays a more prominent role in localization than the other. Mutation of either paxillin-binding site has similar effects on FAK activation and downstream signaling. However, the sites aren't strictly redundant as each mutant exhibits phosphorylation/signaling defects distinct from wild type FAK and a mutant completely defective for paxillin binding. Conclusion The studies demonstrate that the two paxillin-binding sites of FAK are not redundant and that both sites are required for FAK function