Abstract:Since the pioneering work of Hirschfeld, it is known that time-integrated emission (TiEm) of a fluorophore is independent of fluorescence quantum yield and illumination intensity. Practical implementation of this important result for determining exact probe distribution in living cells is often hampered by the presence of autofluorescence. Using kinetic modelling of photobleaching combined with pixel-wise bleach rate fitting of decay models with an updated plugin to the ImageJ program, it is shown that the TiEm of a fluorophore in living cells can be determined exactly from the product of bleaching amplitude and time constant. This applies to mono-exponential bleaching from the first excited singlet and/or triplet state and to multi-exponential combinations of such processes. The TiEm can be used to correct for illumination shading and background autofluorescence without the need for fluorescent test layers or separate imaging of non-stained cells. We apply the method to simulated images and to images of cells, whose membranes were labelled with fluorescent sterols and sphingolipids. Our bleaching model can be extended to include a probability density function (PDF) of intrinsic bleach rate constants with a memory kernel. This approach results in a time-dependent bleach rate coefficient and is exemplified for fluorescent sterols in restricted intracellular environments, like lipid droplets. We show that for small deviations from the classical exponential bleaching, the TiEm of decay functions with rate coefficients remains largely independent of fluorescence lifetime and illumination, and thereby represents a faithful measure of probe distribution.

The endocytic pathway is a complex network of highly dynamic organelles, which has been traditionally studied by quantitative fluorescence microscopy. The data generated by this method can be overwhelming and its analysis, even for the skilled microscopist, is tedious and error-prone. We developed SpatTrack, an open source, platform-independent program collecting a variety of methods for analysis of vesicle dynamics and distribution in living cells. SpatTrack performs Endocytosis of proteins from the cell surface and subsequent sorting of internalized cargo in endosomes is a complex and highly dynamic process. Traditionally, endocytosis has been studied by subcellular fractionation and by transmission electron microscopy after suitable labeling of endocytic ligands (1,2). In parallel, fluorescence microscopy has evolved as a major tool to study the endocytic pathway. This is a consequence of the high specificity and sensitivity of fluorescence and the relative ease of labeling endocytic cargo with small fluorescent probes.Moreover, only fluorescence studies of the endocytic pathway bear the potential of measuring biophysical properties of the endosome, like pH or concentration of ions like calcium and chloride (3,4). Ligands of endocytic receptors have several fates once arrived in the endosome; they can recycle to the cell surface from early sorting or recycling endosomes, like transferrin after releasing iron into the endosomal lumen (5). They can become targeted to the trans-Golgi network in a retrograde trafficking route, as furin or several plant toxins (5,6). Ligands can be also 1406 www.traffic.dk

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Experiences of adversity can generate positive psychological effects alongside negative impacts. Little research to date has evaluated predictors of post-traumatic growth in mental or community healthcare workers during the COVID-19 pandemic. Following a survey of 854 community and mental healthcare staff in the United Kingdom in July to September 2020, multiple linear regression was used to determine the association between hypothesised risk and protective factors (personal, organisational and environmental variables) and total scores on the Post-traumatic Growth Inventory–Short Version. Positive self-reflection activities, black and minority ethnic status, developing new healthcare knowledge and skills, connecting with friends and family, feeling supported by senior management, feeling supported by the UK people, and anxiety about the personal and work-related consequences of COVID-19 each significantly independently predicted greater post-traumatic growth. Working in a clinical role and in mental healthcare or community physical healthcare predicted lower post-traumatic growth. Our research supports the value of taking an organisational growth-focused approach to occupational health during times of adversity, by supporting staff to embrace opportunities for personal growth. Valuing staff’s cultural and religious identity and encouraging self-reflective activities, such as mindfulness and meditation, may help to promote post-traumatic growth.