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Fluorescence microscopy is one of the most indispensable and informative driving forces for biological research, but the extent of observable biological phenomena is essentially determined by the content and quality of the acquired images. To address the different noise sources that can degrade these images, we introduce an algorithm for multiscale image restoration through optimally sparse representation (MIRO). MIRO is a deterministic framework that models the acquisition process and uses pixelwise noise correction to improve image quality. Our study demonstrates that this approach yields a remarkable restoration of the fluorescence signal for a wide range of microscopy systems, regardless of the detector used (e.g., electron-multiplying charge-coupled device, scientific complementary metal-oxide semiconductor, or photomultiplier tube). MIRO improves current imaging capabilities, enabling fast, low-light optical microscopy, accurate image analysis, and robust machine intelligence when integrated with deep neural networks. This expands the range of biological knowledge that can be obtained from fluorescence microscopy.

 

Lipid vesicles immersed in solute gradients are predicted to migrate from regions of high to low solute concentration due to osmotic flows induced across their semipermeable membranes. This process─known as osmophoresis─is potentially relevant to biological processes such as vesicle trafficking and cell migration; however, there exist significant discrepancies (several orders of magnitude) between experimental observations and theoretical predictions for the vesicle speed. Here, we seek to reconcile predictions of osmophoresis with observations of vesicle motion in osmotic gradients. We prepare quasi-steady solute gradients in a microfluidic chamber using density-matched solutions of sucrose and glucose to eliminate buoyancy-driven flows. We quantify the motions of giant DLPC vesicles and Brownian tracer particles in such gradients using Bayesian analysis of particle tracking data. Despite efforts to mitigate convective flows, we observe directed motion of both lipid vesicles and tracer particles in a common direction at comparable speeds of order 10 nm/s. These observations are not inconsistent with models of osmophoresis, which predict slower motion at ca. 1 nm/s; however, experimental uncertainty and the confounding effects of fluid convection prohibit a quantitative comparison. In contrast to previous reports, we find no evidence for anomalously fast osmophoresis of lipid vesicles when fluid convection is mitigated and quantified. We discuss strategies for enhancing the speed of osmophoresis using high permeability membranes and geometric confinement. 

In fluorescence microscopy, the quality of the acquired images determines the extent of observable biological phenomena. To address the different noise sources degrading these images, we introduce a model-based framework compatible with several microscopy systems independently from the detector used.

 

This paper describes mammary organoids with a basal‐in phenotype where the basement membrane is located on the interior surface of the organoid. A key materials consideration to induce this basal‐in phenotype is the use of a minimal gel scaffold that the epithelial cells self‐assemble around and encapsulate. When MDA‐MB‐231 breast cancer cells are co‐cultured with epithelial cells from day 0 under these conditions, cells self‐organize into patterns with distinct cancer cell populations both inside and at the periphery of the epithelial organoid. In another type of experiment, the robust formation of the basement membrane on the epithelial organoid interior enables convenient studies of MDA‐MB‐231 invasion in a tumor progression‐relevant direction relative to epithelial cell‐basement membrane positioning. That is, the study of cancer invasion through the epithelium first, followed by the basement membrane to the basal side, is realized in an experimentally convenient manner where the cancer cells are simply seeded on the outside of preformed organoids, and their invasion into the organoid is monitored. Interestingly, invasion is more prominent when tumor cells are added to day 7 organoids with less developed basement membranes compared to day 16 organoids with more defined ones.