Recovering the Imperfect: Cell Segmentation in the Presence of Dynamically Localized Proteins

Deploying off-the-shelf segmentation networks on biomedical data has become common practice, yet if structures of interest in an image sequence are visible only temporarily, existing frame-by-frame methods fail. In this paper, we provide a solution to segmentation of imperfect data through time based on temporal propagation and uncertainty estimation. We integrate uncertainty estimation into Mask R-CNN network and propagate motion-corrected segmentation masks from frames with low uncertainty to those frames with high uncertainty to handle temporary loss of signal for segmentation. We demonstrate the value of this approach over frame-by-frame segmentation and regular temporal propagation on data from human embryonic kidney (HEK293T) cells transiently transfected with a fluorescent protein that moves in and out of the nucleus over time. The method presented here will empower microscopic experiments aimed at understanding molecular and cellular function.Comment: Accepted at MICCAI Workshop on Medical Image Learning with Less Labels and Imperfect Data, 202

Construction of a Multiwell Light-Induction Platform for Traceless Control of Gene Expression in Mammalian Cells

Mammalian cells can be engineered to incorporate light-responsive elements that reliably sense stimulation by light and activate endogenous pathways, such as the cAMP or Ca2+ pathway, to control gene expression. Light-inducible gene expression systems offer high spatiotemporal resolution, and are also traceless, reversible, tunable, and inexpensive. Melanopsin, a well-known representative of the animal opsins, is a G-protein-coupled receptor that triggers a Gαq-dependent signaling cascade upon activation with blue light (≈470 nm). Here, we describe how to rewire melanopsin activation by blue light to transgene expression in mammalian cells, with detailed instructions for constructing a 96-LED array platform with multiple tunable parameters for illumination of the engineered cells in multiwell plates.ISSN:1064-3745ISSN:1940-602

Optogenetic Techniques for Manipulating and Sensing G Protein-Coupled Receptor Signaling

G protein-coupled receptors (GPCRs) form the largest class of membrane receptors in the mammalian genome with nearly 800 human genes encoding for unique subtypes. Accordingly, GPCR signaling is implicated in nearly all physiological processes. However, GPCRs have been difficult to study due in part to the complexity of their function which can lead to a plethora of converging or diverging downstream effects over different time and length scales. Classic techniques such as pharmacological control, genetic knockout and biochemical assays often lack the precision required to probe the functions of specific GPCR subtypes. Here we describe the rapidly-growing set of optogenetic tools, ranging from methods for optical control of the receptor itself to optical sensing and manipulation of downstream effectors. These tools permit the quantitative measurements of GPCRs and their downstream signaling with high specificity and spatiotemporal precision