2023
DOI: 10.1101/2023.12.18.572148
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RASP: Optimal single fluorescent puncta detection in complex cellular backgrounds

Bin Fu,
Emma E. Brock,
Rebecca Andrews
et al.

Abstract: Super-resolution and single-molecule microscopy are increasingly applied to complex biological systems. A major challenge of this approach 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:… Show more

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Cited by 2 publications
(3 citation statements)
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“…The large size and photosensitivity of these systems make light sheet fluorescence microscopy a useful imaging approach, which comes with well-known challenges of image processing and analysis. Further, these tissues are also often dense and possess strong, structured fluorescence backgrounds that complicate particle detection (Fu et al 2023). Our image analysis pipeline for MS2 spot quantification, along with the plasmids for MCP and MS2 constructs, may be a useful starting point for such endeavors.…”
Section: Discussionmentioning
confidence: 99%
“…The large size and photosensitivity of these systems make light sheet fluorescence microscopy a useful imaging approach, which comes with well-known challenges of image processing and analysis. Further, these tissues are also often dense and possess strong, structured fluorescence backgrounds that complicate particle detection (Fu et al 2023). Our image analysis pipeline for MS2 spot quantification, along with the plasmids for MCP and MS2 constructs, may be a useful starting point for such endeavors.…”
Section: Discussionmentioning
confidence: 99%
“…In brief, the analysis pipeline identifies features in an image, classifies them as either large aggregates or oligomers, and quantifies details such as brightness, size, and position. 45 A detailed description of the analysis is provided in Supplementary Information Note 1: Aggregate-detection pipeline and Supplementary Figures S4-S10. Figure 3a shows a typical PD image containing nano and microscale features.…”
Section: Autofluorescence Suppression and High-sensitivity Microscopy...mentioning
confidence: 99%
“…To perform statistically robust comparisons between samples, we developed a computationally efficient method for detecting and quantifying fluorescent species. This open-source analysis pipeline 45 facilitates the rapid processing of large image libraries, facilitating transparent, shareable, and verifiable results. A schematic illustrating the analysis method and its validation is shown in Figure 3.…”
Section: Autofluorescence Suppression and High-sensitivity Microscopy...mentioning
confidence: 99%