Fluorescent biosensors are important measurement tools for in vivo quantification of pH, concentrations of metal ions and other analytes, and physical parameters such as membrane potential. Both the development of these sensors and their implementation in examining cellular heterogeneity requires technology for measuring and sorting cells based on the fluorescence levels before and after chemical or physical perturbations. We developed a droplet microfluidic platform for the screening and separation of cell populations on the basis of the in vivo response of expressed fluorescence-based biosensors after addition of an exogenous analyte. We demonstrate the capability to resolve the responses of two genetically-encoded Zn2+ sensors at a range of time points spanning several seconds and subsequently sort a mixed-cell population of varying ratios with high accuracy.

Genetically-encoded sensors based on fluorescence resonance energy transfer (FRET) are powerful tools for quantifying and visualizing analytes in living cells, and when targeted to organelles have the potential to define distribution of analytes in different parts of the cell. However, quantitative estimates of analyte distribution require rigorous and systematic analysis of sensor functionality in different locations. In this work, we establish methods to critically evaluate sensor performance in different organelles and carry out a side-by-side comparison of three different genetically encoded sensor platforms for quantifying cellular zinc ions (Zn2+). Calibration conditions are optimized for high dynamic range and stable FRET signals. Using a combination of single-cell microscopy and a novel microfluidic platform capable of screening thousands of cells in a few hours, we observe differential performance of these sensors in the cytosol compared to the ER of HeLa cells, and identify the formation of oxidative oligomers of the sensors in the ER. Finally, we use new methodology to re-evaluate the binding parameters of these sensors both in the test tube and in living cells. Ultimately, we demonstrate that sensor responses can be affected by different cellular environments, and provide a framework for evaluating future generations of organelle-targeted sensors.

Informal physics environments present opportunities for youth to voluntarily engage with physics in a collaborative space. However, individual children's educational backgrounds, expectations, and motivations impact their engagement with the group learning activities and educational tools in informal physics learning. We initiate an investigation into the variables of agency, support, and technology that contribute to children's dynamic epistemic framing of a virtually simulated learning environment within an afterschool physics environment. Specifically, we consider the use of PhET interactive simulations in the PISEC afterschool physics program by middle-school students and their college-aged mentors. We analyzed video of a group of three learners and their mentor as they use a simulation and transition into a physical experiment based on the simulation. We analyzed each individual's actions and vocalizations to identify their framing using a two-dimensional framing axis. Further, we determined when these frames appeared to be aligned or misaligned between group members during the activity, transitions between alignment and misalignment, and investigated what malleable factors (e.g. technology use, mentor pedagogy, program structures) are contributing to these shifts in alignment. Initial analysis shows malleable factors such as UE pedagogy and environment affordances (e.g. technology, spatial orientation), as well as their intersection with non-malleable factors (e.g. youth social motivations) for future investigation.

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