PhagoSight: an open-source MATLAB® package for the analysis of fluorescent neutrophil and macrophage migration in a zebrafish model

Neutrophil migration in zebrafish larvae is increasingly used as a model to study the response of these leukocytes to different determinants of the cellular inflammatory response. However, it remains challenging to extract comprehensive information describing the behaviour of neutrophils from the multi-dimensional data sets acquired with widefield or confocal microscopes. Here, we describe PhagoSight, an open-source software package for the segmentation, tracking and visualisation of migrating phagocytes in three dimensions. The algorithms in PhagoSight extract a large number of measurements that summarise the behaviour of neutrophils, but that could potentially be applied to any moving fluorescent cells. To derive a useful panel of variables quantifying aspects of neutrophil migratory behaviour, and to demonstrate the utility of PhagoSight, we evaluated changes in the volume of migrating neutrophils. Cell volume increased as neutrophils migrated towards the wound region of injured zebrafish. PhagoSight is openly available as MATLAB® m-files under the GNU General Public License. Synthetic data sets and a comprehensive user manual are available from http://www.phagosight.org

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Quantifying the effects of sensor coatings on body temperature measurements

Background: A characterization of an organism's thermoregulatory ability informs our understanding of its physiology, ecology and behavior. Biotelemetry studies on thermoregulation increasingly rely on in situ body temperature measurements from surgically implanted data loggers. To protect the organism and the instrument, the electronics and the temperature sensor are often encased in non-conductive materials prior to insertion into the organism. These materials thermally insulate the sensor, thus potentially biasing temperature measurements to suggest a greater degree of thermoregulation than is actually the case. Results: Here we present methodology to quantify and correct for the effect of sensor coatings on temperature measurements by data recording tags. We illustrate these methods using Wildlife Computer's Mk9 archival tag, field data from the peritoneal cavity of a juvenile albacore tuna (Thunnus alalunga) and simulated data of several species of ectotherms (fish: Hemitripterus americanus, Catostomus commersoni and Maxostoma macrolepidotum; reptiles: Macroclemys temminckii, Varanus spp.), ranging in size from 10 to 1000 g. Mk9 tags had rate constants (measures of the sensor's ability to respond to changes in temperature) of 1.79 ± 0.06 and 0.81 ± 0.07 min-1 for the external and internal sensors, respectively. The higher rate constant of the external sensor produced smaller errors than the internal sensor. Yet, both sensors produced instantaneous errors of over 1 °C for all species tested, with the exception of T. alalunga. Conclusions: The effect of sensor coatings on body temperature measurements is shown to depend on the relative values of the sensor's and the organism's rate constant and the rate of change of environmental temperature. If the sensor's rate constant is lower than that of the organism, the temperature measurements will reflect the thermal properties of the sensor rather than the organism