Investigating dye performance and crosstalk in fluorescence enabled bioimaging using a model system

Detailed imaging of biological structures, often smaller than the diffraction limit, is possible in fluorescence microscopy due to the molecular size and photophysical properties of fluorescent probes. Advances in hardware and multiple providers of high-end bioimaging makes comparing images between studies and between research groups very difficult. Therefore, we suggest a model system to benchmark instrumentation, methods and staining procedures. The system we introduce is based on doped zeolites in stained polyvinyl alcohol (PVA) films: a highly accessible model system which has the properties needed to act as a benchmark in bioimaging experiments. Rather than comparing molecular probes and imaging methods in complicated biological systems, we demonstrate that the model system can emulate this complexity and can be used to probe the effect of concentration, brightness, and cross-talk of fluorophores on the detected fluorescence signal. The described model system comprises of lanthanide (III) ion doped Linde Type A zeolites dispersed in a PVA film stained with fluorophores. We tested: F18, MitoTracker Red and ATTO647N. This model system allowed comparing performance of the fluorophores in experimental conditions. Importantly, we here report considerable cross-talk of the dyes when exchanging excitation and emission settings. Additionally, bleaching was quantified. The proposed model makes it possible to test and benchmark staining procedures before these dyes are applied to more complex biological systems

Expression of telomerase inhibits hydroxyl radical-induced apoptosis in normal telomerase negative human lung fibroblasts

AbstractIn tumor cells telomerase activity is associated with resistance to apoptosis and the introduction of the human telomerase reverse transcriptase (hTERT) subunit into normal human cells is associated with life span extension of the cells. To determine the role of telomerase in regulating apoptosis, telomerase negative human embryo lung fibroblasts were transfected with the hTERT gene. Unlike the control fibroblasts, the telomerase-expressing cells had elongated telomeres and were resistant to apoptosis induced by hydroxyl radicals. The results indicate that expression of telomerase and, thus, the maintenance of telomere length in normal human somatic cells caused resistance to not only cellular senescence but also apoptosis. Moreover, we found that hydroxyl radical-induced apoptosis in telomerase-expressing and control fibroblasts was caspase-3 independent. These findings have revealed a new type of interrelation between telomerase and caspase-3, which may indicate that in this case the expressed telomerase may inhibit apoptosis at a site not related to the caspase-3 cascade