Images in fluorescence microscopy are inherently blurred due to the limit of diffraction of light. The purpose of deconvolution microscopy is to compensate numerically for this degradation. Deconvolution is widely used to restore fine details of 3D biological samples. Unfortunately, dealing with deconvolution tools is not straightforward. Among others, end users have to select the appropriate algorithm, calibration and parametrization, while potentially facing demanding computational tasks. To make deconvolution more accessible, we have developed a practical platform for deconvolution microscopy called DeconvolutionLab. Freely distributed, DeconvolutionLab hosts standard algorithms for 3D microscopy deconvolution and drives them through a user-oriented interface. In this paper, we take advantage of the release of DeconvolutionLab2 to provide a complete description of the software package and its built-in deconvolution algorithms. We examine several standard algorithms used in deconvolution microscopy, notably: Regularized inverse filter, Tikhonov regularization, Landweber, Tikhonov-Miller, Richardson-Lucy, and fast iterative shrinkage-thresholding. We evaluate these methods over large 3D microscopy images using simulated datasets and real experimental images. We distinguish the algorithms in terms of image quality, performance, usability and computational requirements. Our presentation is completed with a discussion of recent trends in deconvolution, inspired by the results of the Grand Challenge on deconvolution microscopy that was recently organized.

Motor proteins and microtubule-associated proteins (MAPs) play important roles in cellular transport, regulation of shape and polarity of cells. While motor proteins generate motility, MAPs are thought to stabilize the microtubule tracks. However, the proteins also interfere with each other, such that MAPs are able to inhibit transport of vesicles and organelles in cells. In order to investigate the mechanism of MAP±motor interference in molecular detail, we have studied single kinesin molecules by total internal re¯ection¯uorescence microscopy in the presence of different neuronal MAPs (tau, MAP2c). The parameters observed included run-length (a measure of processivity), velocity and frequency of attachment. The main effect of MAPs was to reduce the attachment frequency of motors. This effect was dependent on the concentration, the af®nity to microtubules and the domain composition of MAPs. In contrast, once attached, the motors did not show a change in speed, nor in their run-length. The results suggest that MAPs can regulate motor activity on the level of initial attachment, but not during motion.

Approximately one-third of the world's population suffers from chronic helminth infections with no effective vaccines currently available. Antibodies and alternatively activated macrophages (AAM) form crucial components of protective immunity against challenge infections with intestinal helminths. However, the mechanisms by which antibodies target these large multi-cellular parasites remain obscure. Alternative activation of macrophages during helminth infection has been linked to signaling through the IL-4 receptor alpha chain (IL-4Rα), but the potential effects of antibodies on macrophage differentiation have not been explored. We demonstrate that helminth-specific antibodies induce the rapid trapping of tissue migrating helminth larvae and prevent tissue necrosis following challenge infection with the natural murine parasite Heligmosomoides polygyrus bakeri (Hp). Mice lacking antibodies (JH −/−) or activating Fc receptors (FcRγ−/−) harbored highly motile larvae, developed extensive tissue damage and accumulated less Arginase-1 expressing macrophages around the larvae. Moreover, Hp-specific antibodies induced FcRγ- and complement-dependent adherence of macrophages to larvae in vitro, resulting in complete larval immobilization. Antibodies together with helminth larvae reprogrammed macrophages to express wound-healing associated genes, including Arginase-1, and the Arginase-1 product L-ornithine directly impaired larval motility. Antibody-induced expression of Arginase-1 in vitro and in vivo occurred independently of IL-4Rα signaling. In summary, we present a novel IL-4Rα-independent mechanism of alternative macrophage activation that is antibody-dependent and which both mediates anti-helminth immunity and prevents tissue disruption caused by migrating larvae.

Kinesin-1 is a processive molecular motor transporting cargo along microtubules. Inside cells, several motors and microtubule-associated proteins compete for binding to microtubules. Therefore, the question arises how processive movement of kinesin-1 is affected by crowding on the microtubule. Here we use total internal reflection fluorescence microscopy to image in vitro the runs of single quantum dot-labelled kinesins on crowded microtubules under steady-state conditions and to measure the degree of crowding on a microtubule at steady-state. We find that the runs of kinesins are little affected by high kinesin densities on a microtubule. However, the presence of high densities of a mutant kinesin that is not able to step efficiently reduces the average speed of wild-type kinesin, while hardly changing its processivity. This indicates that kinesin waits in a strongly bound state on the microtubule when encountering an obstacle until the obstacle unbinds and frees the binding site for kinesin's next step. A simple kinetic model can explain quantitatively the behaviour of kinesin under both crowding conditions.

The formin Fus1 nucleates a novel actin structure in fission yeast, named the actin fusion focus, which consists of an aster of actin filaments whose barbed ends are focalized at a membrane proximal site and serves to focalize cell wall hydrolase delivery for cell fusion.