High-throughput single-cell transcriptomics offers an unbiased approach for understanding the extent, basis, and function of gene expression variation between seemingly identical cells. Here, we sequence single-cell RNA-Seq libraries prepared from over 1,700 primary mouse bone marrow derived dendritic cells (DCs) spanning several experimental conditions. We find substantial variation between identically stimulated DCs, in both the fraction of cells detectably expressing a given mRNA and the transcript’s level within expressing cells. Distinct gene modules are characterized by different temporal heterogeneity profiles. In particular, a “core” module of antiviral genes is expressed very early by a few “precocious” cells, but is later activated in all cells. By stimulating cells individually in sealed microfluidic chambers, analyzing DCs from knockout mice, and modulating secretion and extracellular signaling, we show that this response is coordinated via interferon-mediated paracrine signaling. Surprisingly, preventing cell-to-cell communication also substantially reduces variability in the expression of an early-induced “peaked” inflammatory module, suggesting that paracrine signaling additionally represses part of the inflammatory program. Our study highlights the importance of cell-to-cell communication in controlling cellular heterogeneity and reveals general strategies that multicellular populations use to establish complex dynamic responses.

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Leading edge technologies require continually shrinking design grids due to the demands of decreasing minimum feature size and higher resolution. Using conventional raster-scanned exposure tools to place these patterns on photomasks results in longer write times, because linear decreases in address result in exponential increases in write time. This phenomenon can be compensated for by changes in writing strategies. Multipass gray (MPG) is one method of drastically improving throughput at small addresses while retaining lithographic quality.MPG was introduced with the MEBES 4500S system and takes advantage of the gray-level writing strategies of the CORE® and ALTA® exposure systems. MPG combines the use of multiple exposure passes and offset scan voting to reduce placement, butting, and scan linearity errors. Additionally, the multipass strategy allows the exposure of higher doses without impacting throughput. The added dose allows use of less sensitive resists such as DNQ/novolacs and high-molecular weight, chain scission resists such as ZEP 7000. This paper describes the MPG writing strategy and presents lithographic results. Results are based on a process using GHOST proximity effect correction (PEC), ZEP 7000 resist, and dry etch of the chrome film. Placement and butting error reduction with multipass writing, critical dimension (CD) control, CD linearity, and overall lithographic quality will be discussed. MULTIPASS GRAY WRITING STRATEGYRaster-scan writing techniques have evolved through several stages, beginning with simple single-pass printing (SPP). The latest stage in this evolution for MEBES systems, MPG, is built on the successful implementation of gray-level writing strategies for the CORE and ALTA laser systems. As pattern addresses become smaller with each device generation, the writing time of systems operating with SPP becomes impractical, because the writing time increases quadratically with linear decreases in address size. Compared to writing equivalent patterns with SPP, MPG delivers four times the dose with approximately four times the throughput. Notably, at small address sizes, CD quality is not lost, and often it is better than that obtained with SPP. Combined with GHOST PEC, 180-nm devices can be generated with good CD uniformity and linearity.To understand how the MPG process can produce such detailed features with high throughput and dose, a brief review of writing strategies is in order. SPP is the original writing mode for MEBES systems, and it is the most intuitive. Analogous to single-pass scanning in a cathode ray tube, the electron-beam (e-beam) is scanned in raster fashion across the entire quality area of the plate. The full-width, half-maximum of the beam is, nominally, equal to the grid spacing in the raster. Given a sharply focused beam, SPP can produce detailed features, but with unacceptable time penalties at small addresses. In addition, minor, normal perturbations of beam quality during the single scan negatively impact CD control and uniformity.Multipass printing (2X MPP...

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