RASP: Optimal Single Puncta Detection in Complex Cellular Backgrounds.

Super-resolution and single-molecule microscopies have been increasingly applied to complex biological systems. A major challenge of these approaches is that fluorescent puncta must be detected in the low signal, high noise, heterogeneous background environments of cells and tissue. We present RASP, Radiality Analysis of Single Puncta, a bioimaging-segmentation method that solves this problem. RASP removes false-positive puncta that other analysis methods detect and detects features over a broad range of spatial scales: from single proteins to complex cell phenotypes. RASP outperforms the state-of-the-art methods in precision and speed using image gradients to separate Gaussian-shaped objects from the background. We demonstrate RASP's power by showing that it can extract spatial correlations between microglia, neurons, and α-synuclein oligomers in the human brain. This sensitive, computationally efficient approach enables fluorescent puncta and cellular features to be distinguished in cellular and tissue environments, with sensitivity down to the level of the single protein. Python and MATLAB codes, enabling users to perform this RASP analysis on their own data, are provided as Supporting Information and links to third-party repositories.

Real-time 3D tracking of swimming microbes using digital holographic microscopy and deep learning.

The three-dimensional swimming tracks of motile microorganisms can be used to identify their species, which holds promise for the rapid identification of bacterial pathogens. The tracks also provide detailed information on the cells' responses to external stimuli such as chemical gradients and physical objects. Digital holographic microscopy (DHM) is a well-established, but computationally intensive method for obtaining three-dimensional cell tracks from video microscopy data. We demonstrate that a common neural network (NN) accelerates the analysis of holographic data by an order of magnitude, enabling its use on single-board computers and in real time. We establish a heuristic relationship between the distance of a cell from the focal plane and the size of the bounding box assigned to it by the NN, allowing us to rapidly localise cells in three dimensions as they swim. This technique opens the possibility of providing real-time feedback in experiments, for example by monitoring and adapting the supply of nutrients to a microbial bioreactor in response to changes in the swimming phenotype of microbes, or for rapid identification of bacterial pathogens in drinking water or clinical samples.