Polymer Patterning by Laser-Induced Multipoint Initiation of Frontal Polymerization.

Frontal polymerization (FP) is an approach for thermosetting plastics at a lower energy cost than an autoclave. The potential to generate simultaneous propagation of multiple polymerization fronts has been discussed as an exciting possibility. However, FP initiated at more than two points simultaneously has not been demonstrated. Multipoint initiation could enable both large-scale material fabrication and unique pattern generation. Here, the authors present laser-patterned photothermal heating as a method for simultaneous initiation of FP at multiple locations in a 2-D sample. Carbon black particles are mixed into liquid resin (dicyclopentadiene) to enhance absorption of light from a Ti:sapphire laser (800 nm) focused on a sample. The laser is time-shared by rapid steering among initiation points, generating polymerization using up to seven simultaneous points of initiation. This process results in the formation of both symmetric and asymmetric seam patterns resulting from the collision of fronts. The authors also present and validate a theoretical framework for predicting the seam patterns formed by front collisions. This framework allows the design of novel patterns via an inverse solution for determining the initiation points required to form a desired pattern. Future applications of this approach could enable rapid, energy-efficient manufacturing of novel composite-like patterned materials.

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization.

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an application of smFISH to measure the rates of transcription and mRNA degradation in Escherichia coli. As smFISH is based on fixed cells, we perform smFISH at multiple time points during a time-course experiment, i.e., when cells are undergoing synchronized changes upon induction or repression of gene expression. At each time point, sub-regions of an mRNA are spectrally distinguished to probe transcription elongation and premature termination. The outcome of this protocol also allows for analyzing intracellular localization of mRNAs and heterogeneity in mRNA copy numbers among cells. Using this protocol many samples (~50) can be processed within 8 h, like the amount of time needed for just a few samples. We discuss how to apply this protocol to study the transcription and degradation kinetics of different mRNAs in bacterial cells.