Two-photon excitation selective plane illumination microscopy (2PE-SPIM) of highly scattering samples: Characterization and application

In this work we report the advantages provided by two photon excitation (2PE) implemented in a selective plane illumination microscopy (SPIM) when imaging thick scattering samples. In particular, a detailed analysis of the effects induced on the real light sheet excitation intensity distribution is performed. The comparison between single-photon and twophoton excitation profiles shows the reduction of the scattering effects and sample-induced aberrations provided by 2PE-SPIM. Furthermore, uniformity of the excitation distribution and the consequent improved image contrast is shown when imaging scattering phantom samples in depth by 2PE-SPIM. These results show the advantages of 2PE-SPIM and suggest how this combination can further enhance the SPIM performance. Phantom samples have been designed with optical properties compatible with biological applications of interest. © 2013 Optical Society of America

The dark recovery rate in the photocycle of the bacterial photoreceptor YtvA is affected by the cellular environment and by hydration

We report thermal recovery kinetics of the lit state into the parental dark state, measured for the blue light-sensing photoreceptor YtvA inside overexpressing E. coli and B. subtilis bacterial cells, performed for the wild type and several mutated proteins. Recovery was followed as a recovery of the fluorescence, as this property is only found for the parental but not for the photochemically generated lit state. When cells were deposited onto a microscope glass plate, the observed thermal recovery rate in the photocycle was found ca. ten times faster in comparison to purified YtvA in solution. When the E. coli or B. subtilis colonies were soaked in an isotonic buffer, the dark relaxation became again much slower and was very similar to that observed for YtvA in solution. The observed effects show that rate constants can be tuned by the cellular environment through factors such as hydration. Copyright

Subnuclear localization, rates and effectiveness of UVC-induced unscheduled DNA synthesis visualized by fluorescence widefield, confocal and super-resolution microscopy

Unscheduled DNA synthesis (UDS) is the final stage of the process of repair of DNA lesions induced by UVC. We detected UDS using a DNA precursor, 5-ethynyl-2′-deoxyuridine (EdU). Using wide-field, confocal and super-resolution fluorescence microscopy and normal human fibroblasts, derived from healthy subjects, we demonstrate that the sub-nuclear pattern of UDS detected via incorporation of EdU is different from that when BrdU is used as DNA precursor. EdU incorporation occurs evenly throughout chromatin, as opposed to just a few small and large repair foci detected by BrdU. We attribute this difference to the fact that BrdU antibody is of much larger size than EdU, and its accessibility to the incorporated precursor requires the presence of denatured sections of DNA. It appears that under the standard conditions of immunocytochemical detection of BrdU only fragments of DNA of various length are being denatured. We argue that, compared with BrdU, the UDS pattern visualized by EdU constitutes a more faithful representation of sub-nuclear distribution of the final stage of nucleotide excision repair induced by UVC. Using the optimized integrated EdU detection procedure we also measured the relative amount of the DNA precursor incorporated by cells during UDS following exposure to various doses of UVC. Also described is the high degree of heterogeneity in terms of the UVC-induced EdU incorporation per cell, presumably reflecting various DNA repair efficiencies or differences in the level of endogenous dT competing with EdU within a population of normal human fibroblasts

Unsupervised Spike Sorting for Large-Scale, High-Density Multielectrode Arrays

We present a method for automated spike sorting for recordings with high-density, large-scale multielectrode arrays. Exploiting the dense sampling of single neurons by multiple electrodes, an efficient, low-dimensional representation of detected spikes consisting of estimated spatial spike locations and dominant spike shape features is exploited for fast and reliable clustering into single units. Millions of events can be sorted in minutes, and the method is parallelized and scales better than quadratically with the number of detected spikes. Performance is demonstrated using recordings with a 4,096-channel array and validated using anatomical imaging, optogenetic stimulation, and model-based quality control. A comparison with semi-automated, shape-based spike sorting exposes significant limitations of conventional methods. Our approach demonstrates that it is feasible to reliably isolate the activity of up to thousands of neurons and that dense, multi-channel probes substantially aid reliable spike sorting

Super-Resolution Imaging Strategies for Cell Biologists Using a Spinning Disk Microscope

In this study we use a spinning disk confocal microscope (SD) to generate super-resolution images of multiple cellular features from any plane in the cell. We obtain super-resolution images by using stochastic intensity fluctuations of biological probes, combining Photoactivation Light-Microscopy (PALM)/Stochastic Optical Reconstruction Microscopy (STORM) methodologies. We compared different image analysis algorithms for processing super-resolution data to identify the most suitable for analysis of particular cell structures. SOFI was chosen for X and Y and was able to achieve a resolution of ca. 80 nm; however higher resolution was possible >30 nm, dependant on the super-resolution image analysis algorithm used. Our method uses low laser power and fluorescent probes which are available either commercially or through the scientific community, and therefore it is gentle enough for biological imaging. Through comparative studies with structured illumination microscopy (SIM) and widefield epifluorescence imaging we identified that our methodology was advantageous for imaging cellular structures which are not immediately at the cell-substrate interface, which include the nuclear architecture and mitochondria. We have shown that it was possible to obtain two coloured images, which highlights the potential this technique has for high-content screening, imaging of multiple epitopes and live cell imaging