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Data Requirements

NeuronStudio was designed to be used with datasets obtained by confocal or multi-photon fluorescence microscopy. The program supports files in TIFF (Tagged Image File Format) and RAW (binary) format. Currently the program only accepts greyscale images of 8 or 16 bit-depth. 12-bit greyscale is also allowed but each pixel must be padded to 16 bits (i.e. 4 unused bits). The data obtained from these systems is of varying quality depending on the imaging parameters used. In general, the optimal resolution is dictated by the optics of the equipment as they relate to the Nyquist rate. Selecting the axial and lateral resolutions this way may yield highly asymmetrical voxels so it is recommended that the asymmetry be reduced even if that means over-sampling somewhat along the optical axis. A step size that is twice the lateral voxel size, or smaller, is usually considered appropriate but cubic voxels are desirable (storage permitting).

It is also good practice to adjust the exposure to limit the number of over-exposed or saturated voxels along the structure while at the same time limiting the number of under-exposed or black voxels in the background. These adjustments should be performed after any frame or line averaging. This is true for both 8-bit and 12-bit data as it ensures the proper utilization of the dynamic range of the camera.

One way to improve the Signal-to-Noise Ratio (SNR) of the data is to process the dataset with deconvolution software. This not only removes some of the shot noise introduced by the CCD, but most importantly removes some of the blur introduced by the microscope's optics. Deconvolution improves the optical sectioning created by the microscope and allows better discretization of morphologic features, as well as more accurate measurements.

For coverage of large portions of a dendritic structure at high resolution, you may need to acquire multiple stacks at consecutive neighboring locations along the structure and join them together. This can be automated if your microscope has a programmable motorized XY stage. Otherwise this can be done manually, provided that some overlap exists in the stacks, using the VIAS tool provided at our website

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