A limited number of signaling pathways are repeatedly used to regulate a wide variety of processes during development and differentiation. The lack of tools to manipulate signaling pathways dynamically in space and time has been a major technical challenge for biologists. Optogenetic techniques, which utilize light to control protein functions in a reversible fashion, hold promise for modulating intracellular signaling networks with high spatial and temporal resolution. Applications of optogenetics in multicellular organisms, however, have not been widely reported. Here, we create an optimized bicistronic optogenetic system using Arabidopsis thaliana cryptochrome 2 (CRY2) protein and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). In a proofof-principle study, we develop an optogenetic Raf kinase that allows reversible light-controlled activation of the Raf/MEK/ERK signaling cascade. In PC12 cells, this system significantly improves lightinduced cell differentiation compared with co-transfection. When applied to Xenopus embryos, this system enables blue lightdependent reversible Raf activation at any desired developmental stage in specific cell lineages. Our system offers a powerful optogenetic tool suitable for manipulation of signaling pathways with high spatial and temporal resolution in a wide range of experimental settings.

Carbon dots (CDs) are extensively studied to investigate their unique optical properties such as undergoing electron transfer in different scenarios. This study presents an in-depth investigation to study the ensemble-averaged state/bulk state and single-particle level photophysical properties of CDs that have been passivated with electron-accepting (CD-A) and electron-donating molecules (CD-D) on their surface. Our ensemble-averaged state experiments including UV-Vis absorbance titrations, time-resolved photoluminescence (PL) spectra, and 2D emission studies depicted that both CD-A or CD-D had a blue-shift in emission, with a drastic increase in emission intensity, and consequently higher quantum yields, and that acceptor populations (CD-A) dominate overall photophysical properties. Interestingly, transmission electron microscopy and atomic force microscopy revealed that the mixing of donor and acceptor particles (CD-A+ CD-D) leads to a formation of at least two associated geometries, which was dependent on time, concentration, intramolecular electron/charge transfer and hydrogen bonding. On the other hand, single-particle studies revealed that the instantaneous intensity of CDs was comparable, but that CD-A and CD-D have a larger on-time duty cycle, attributed to an increase of blinking frequency. On-and off-time power-law analysis further revealed that CD-A has a larger off-time distribution slope than bare CDs, This article is protected by copyright. All rights reserved. 3 while their on-time distribution is similar. CD-D exhibits an increase in both on-and off-time distribution slope compared to bare CDs. These results indicate that the electron donor provides additional bright and dark states, while the electron acceptor primarily provides bright states, which can explain the increased blinking frequency in CD-A and CD-D at the single-particle level. Singleparticle studies, however, did not reveal an "acceptor-dominating" scenario based on analysis of instantaneous intensity, bleaching kinetics, and photoblinking, indicating that the direct interaction of CD-A and CD-D may affect their photophysical properties in the bulk state due to formation of hierarchical structural assemblies. We anticipate that these fundamental results will further provide insights towards our understanding of the complex mechanism associated with CD emission, which is one of the key contributors to their successful application. As an immediate application of these CDs, we have shown that they can be used as a sensing array for metal ions and can serve as a powerful toolbox for the technological application of CDs.

Highlights d Non-neuronal optogenetics allows for the control of receptor tyrosine kinase activity d Tyrosine 490 of TrkA directly activates the ERK signaling pathway d Tyrosine 785 of TrkA activates ERK signaling through the PLCg-PKC pathway d Tyrosines 490 and 785 of TrkA both contribute to PC12 cell differentiation

For robust single particle optical detection, a high sensitivity in photoluminescence (PL) of Carbon Dots (CDs) must be achieved. PL sensitivity can be successfully correlated with their surface chemistry but requires high synthetic control without altering their basic surface properties. Here we describe conditions for the controlled synthesis of CDs that resulted in a PL sensitivity at the single-particle level. We report that a stoichiometric catalyst N-methyl morpholine-N-oxide (NMMO) can be used as a 'sacrificial' single additive to aid nanoscale surface oxidation. A 24 h NMMO-mediated oxidation increased coverage of oxidized nanoscale surface 3% to 20.9%. NMMO-oxidized CDs (CD-NMMOs) display superior particle brightness, as evidenced by the increase of light absorbance and an enhancement of quantum yield which is characterized by a series of physicochemical and biophysical experiments. We also demonstrate that CD-NMMOs is well suited for intracellular and single-particle imaging.