Light-sheet fluorescence microscopy (LSFM), a prominent fluorescence microscopy technique, offers enhanced temporal resolution for imaging biological samples in four dimensions (4D;x, y, z, time). Some of the most rec...
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Light-sheet fluorescence microscopy (LSFM), a prominent fluorescence microscopy technique, offers enhanced temporal resolution for imaging biological samples in four dimensions (4D;x, y, z, time). Some of the most recent implementations, including inverted selective plane illumination microscopy (iSPIM) and lattice light-sheet microscopy (LLSM), move the sample substrate at an oblique angle relative to the detection objective's optical axis. data from such tilted-sample-scan LSFMs require subsequent deskewing and rotation for proper visualisation and analysis. Such data preprocessing operations currently demand substantial memory allocation and pose significant computational challenges for large 4D dataset. The consequence is prolonged data preprocessing time compared to data acquisition time, which limits the ability for live-viewing the data as it is being captured by the microscope. To enable the fast preprocessing of large light-sheet microscopydatasets without significant hardware demand, we have developed WH-Transform, a memory-efficient transformation algorithm for deskewing and rotating the raw dataset, significantly reducing memory usage and the run time by more than 10-fold for large image stacks. Benchmarked against the conventional method and existing software, our approach demonstrates linear runtime compared to the cubic and quadratic runtime of the other approaches. Preprocessing a raw 3D volume of 2 GB (512 x 1536 x 600 pixels) can be accomplished in 3 s using a GPU with 24 GB of memory on a single workstation. Applied to 4D LLSM datasets of human hepatocytes, lung organoid tissue and brain organoid tissue, our method provided rapid and accurate preprocessing within seconds. Importantly, such preprocessing speeds now allow visualisation of the raw microscope data stream in real time, significantly improving the usability of LLSM in biology. In summary, this advancement holds transformative potential for light-sheet microscopy, enabling real-time, on
A method is presented to determine the feature resolution of physically relevant metrics of data obtained from segmented image sets. The presented method determines the best-fit distribution curve of a dataset by anal...
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A method is presented to determine the feature resolution of physically relevant metrics of data obtained from segmented image sets. The presented method determines the best-fit distribution curve of a dataset by analyzing a truncated portion of the data. An effective resolvable size for the metric of interest is established when including parts of the truncated dataset results in exceeding a specified error tolerance. As such, this method allows for the determination of the feature resolution regardless of the processing parameters or imaging instrumentation. Additionally, the number of missing objects that exist below the resolution of the instrumentation may be estimated. The application of the developed method was demonstrated on data obtained via 2D scanning electron microscopy of a pressed explosive material and from 3D micro X-ray computed tomography of a polymer-bonded explosive material. It was shown that the minimum number of pixels/voxels required for the accurate determination of a physically relevant metric is dependent on the metric of interest. This proposed method, utilizing the prior knowledge of the distribution of metrics of interest, was found to be well suited to determine the feature resolution in applications where large datasets can be achieved.
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