Elric Esposito

In recent years, multielectrode arrays and large silicon probes have been developed to record simultaneously between hundreds and thousands of electrodes packed with a high density. However, they require novel methods to extract the spiking activity of large ensembles of neurons. Here, we developed a new toolbox to sort spikes from these large-scale extracellular data. To validate our method, we performed simultaneous extracellular and loose patch recordings in rodents to obtain 'ground truth' data, where the solution to this sorting problem is known for one cell. The performance of our algorithm was always close to the best expected performance, over a broad range of signal-to-noise ratios, in vitro and in vivo. The algorithm is entirely parallelized and has been successfully tested on recordings with up to 4225 electrodes. Our toolbox thus offers a generic solution to sort accurately spikes for up to thousands of electrodes.

We report the creation of white-light, achromatic Bessel beams using a spatial light modulator and a prism to compensate for the dispersion. Unlike the Bessel beam created by a refractive axicon, this achromatic beam has a radial wavevector and hence an intensity cross-section which is independent of wavelength. The technique also lends itself to the generation of higher order Bessel beams with an on-axis optical vortex and associated orbital angular momentum.

BACKGROUND: Quantitative imaging of epithelial tissues requires bioimage analysis tools that are widely applicable and accurate. In the case of imaging 3D tissues, a common preprocessing step consists of projecting the acquired 3D volume on a 2D plane mapping the tissue surface. While segmenting the tissue cells is amenable on 2D projections, it is still very difficult and cumbersome in 3D. However, for many specimen and models used in developmental and cell biology, the complex content of the image volume surrounding the epithelium in a tissue often reduces the visibility of the biological object in the projection, compromising its subsequent analysis. In addition, the projection may distort the geometry of the tissue and can lead to strong artifacts in the morphology measurement. RESULTS: Here we introduce a user-friendly toolbox built to robustly project epithelia on their 2D surface from 3D volumes and to produce accurate morphology measurement corrected for the projection distortion, even for very curved tissues. Our toolbox is built upon two components. LocalZProjector is a configurable Fiji plugin that generates 2D projections and height-maps from potentially large 3D stacks (larger than 40 GB per time-point) by only incorporating signal of the planes with local highest variance/mean intensity, despite a possibly complex image content. DeProj is a MATLAB tool that generates correct morphology measurements by combining the height-map output (such as the one offered by LocalZProjector) and the results of a cell segmentation on the 2D projection, hence effectively deprojecting the 2D segmentation in 3D. In this paper, we demonstrate their effectiveness over a wide range of different biological samples. We then compare its performance and accuracy against similar existing tools. CONCLUSIONS: We find that LocalZProjector performs well even in situations where the volume to project also contains unwanted signal in other layers. We show that it can process large images without a pre-processing step. We study the impact of geometrical distortions on morphological measurements induced by the projection. We measured very large distortions which are then corrected by DeProj, providing accurate outputs.

Faithful reporting of temporal patterns of intracellular Ca(2+) dynamics requires the working range of indicators to match the signals. Current genetically encoded calmodulin-based fluorescent indicators are likely to distort fast Ca(2+) signals by apparent saturation and integration due to their limiting fluorescence rise and decay kinetics. A series of probes was engineered with a range of Ca(2+) affinities and accelerated kinetics by weakening the Ca(2+)-calmodulin-peptide interactions. At 37 °C, the GCaMP3-derived probe termed GCaMP3fast is 40-fold faster than GCaMP3 with Ca(2+) decay and rise times, t1/2, of 3.3 ms and 0.9 ms, respectively, making it the fastest to-date. GCaMP3fast revealed discreet transients with significantly faster Ca(2+) dynamics in neonatal cardiac myocytes than GCaMP6f. With 5-fold increased two-photon fluorescence cross-section for Ca(2+) at 940 nm, GCaMP3fast is suitable for deep tissue studies. The green fluorescent protein serves as a reporter providing important novel insights into the kinetic mechanism of target recognition by calmodulin. Our strategy to match the probe to the signal by tuning the affinity and hence the Ca(2+) kinetics of the indicator is applicable to the emerging new generations of calmodulin-based probes.