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Cell differentiation is an essential biological process that is often subject to strict temporal regulation. The aquatic bacterium,Caulobacter crescentus, undergoes obligate differentiation from a swarmer cell to a stationary, replication-competent stalked cell, with each cell cycle. Here, we report that the switch from phosphatase to kinase activity of the histidine kinase PleC contributes to timing this differentiation event. We show that PleC PAS domain interaction with the polar scaffold protein PodJ localizes PleC to the cell pole and inhibitsin vivokinase activity. Upon PodJ degradation, released PleC switches to its kinase form and phosphorylates the PleD diguanylate cyclase, initiating the signaling pathway responsible for differentiation. While PodJ inhibits PleC kinase activity, it does not impact PleC phosphatase activity on DivK, which is required for pili biogenesis and flagellar rotation. Thus, PleC PAS domain interaction with PodJ regulates PleC subcellular localization, enzymatic activity, and the timing of cell differentiation, revealing that PAS domains affect enzymatic function on diverse substrates by relying on context dependent binding partners.

Significance The correlation of cryogenic superresolution fluorescence imaging, which features single-molecule sensitivity and specific labelling, with the molecular-scale cellular context of cryogenic electron tomography is a powerful approach for observing the subcellular arrangement of biomolecules. This approach is still in its infancy, and numerous challenges need to be overcome to make this approach routine. This manuscript describes a four-stage workflow using cryogenic single-molecule localizations to annotate cryogenic electron tomography images. The workflow is applied to three different proteins in Caulobacter crescentus and is used to visualize the relative subcellular organization of two proteins, SpmX and PopZ, whose interactions play a key regulatory role in the life cycle of C. crescentus., Superresolution fluorescence microscopy and cryogenic electron tomography (CET) are powerful imaging methods for exploring the subcellular organization of biomolecules. Superresolution fluorescence microscopy based on covalent labeling highlights specific proteins and has sufficient sensitivity to observe single fluorescent molecules, but the reconstructions lack detailed cellular context. CET has molecular-scale resolution but lacks specific and nonperturbative intracellular labeling techniques. Here, we describe an imaging scheme that correlates cryogenic single-molecule fluorescence localizations with CET reconstructions. Our approach achieves single-molecule localizations with an average lateral precision of 9 nm, and a relative registration error between the set of localizations and CET reconstruction of ∼30 nm. We illustrate the workflow by annotating the positions of three proteins in the bacterium Caulobacter crescentus: McpA, PopZ, and SpmX. McpA, which forms a part of the chemoreceptor array, acts as a validation structure by being visible under both imaging modalities. In contrast, PopZ and SpmX cannot be directly identified in CET. While not directly discernable, PopZ fills a region at the cell poles that is devoid of electron-dense ribosomes. We annotate the position of PopZ with single-molecule localizations and confirm its position within the ribosome excluded region. We further use the locations of PopZ to provide context for localizations of SpmX, a low-copy integral membrane protein sequestered by PopZ as part of a signaling pathway that leads to an asymmetric cell division. Our correlative approach reveals that SpmX localizes along one side of the cell pole and its extent closely matches that of the PopZ region.

Biomolecular condensates formed via phase separation enable spatial and temporal organization of enzyme activity. The emergent properties of many condensates have been shown to be responsive to intracellular Adenosine triphosphate (ATP) levels, although the consequences of such mechanisms on enzyme activity are unknown. Here, we show that ATP depletion promotes phase separation in condensates composed of a disordered protein, thereby enhancing the activity of a client kinase enabling robust signaling and maintenance of viability under the stress posed by nutrient scarcity. We propose that a diverse repertoire of condensates can serve as control knobs to tune multivalency and reactivity in response to the metabolic state of bacterial cells.One Sentence SummaryBacterial condensates boost kinase activity under ATP depletion

We asked young scientists to serve as peer mentors for “Seeking Career Clarity,” the author of the question below. By asking refl ective questions, sharing relevant personal experiences, and of ering advice, these scientists provide support and perspective. Follow NextGen Voices on Twitter with hashtag #NextGenSci.

Single-molecule super-resolution fluorescence microscopy conducted in vitrified samples at cryogenic temperatures offers enhanced localization precision due to reduced photobleaching rates, a chemical-free and rapid fixation method, and the potential of correlation with cryogenic electron microscopy. Achieving cryogenic super-resolution microscopy requires the ability to control the sparsity of emissive labels at cryogenic temperatures. Obtaining this control presents a key challenge for the development of this technique. In this work, we identify a red photoactivatable protein, PAmKate, which remains activatable at cryogenic temperatures. We characterize its activation as a function of temperature and find that activation is efficient at cryogenic and room temperatures. We perform cryogenic super-resolution experiments in situ, labeling PopZ, a protein known to assemble into a microdomain at the poles of the model bacterium Caulobacter crescentus. We find improved localization precision at cryogenic temperatures compared to room temperature by a factor of 4, attributable to reduced photobleaching.

Fluorescent protein based pH sensors are a useful tool for measuring protein trafficking through pH changes associated with endo-and exocytosis. However, commonly used pH sensing probes are ubiquitously expressed with their protein of interest throughout the cell, hindering the ability to focus on specific trafficking pools of proteins. We developed a family of excitation-ratiometric, activatable pH responsive tandem dyes, consisting of a pH sensitive Cy3 donor linked to a fluorogenic malachite green acceptor. These cell-excluded dyes are targeted and activated upon binding to a genetically expressed fluorogen activating protein, and are suitable for selective labeling of surface proteins for analysis of endocytosis and recycling in live cells using both confocal and superresolution microscopy. Quantitative profiling of the endocytosis and recycling of tagged β2-adrenergic receptor (B2AR) at a single vesicle level revealed differences among B2AR agonists, consistent with more detailed pharmacological profiling.

Single-molecule superresolution methods enable imaging of specifically-labeled biological samples with structures on length scales below the diffraction limit of visible light. Imaging samples at cryogenic temperatures (77 K) significantly reduces photobleaching, allowing more photons to be collected per emitter and thus improving the localization precision. Cryogenic single-molecule imaging also facilitates correlative imaging with cryogenic electron tomography (cryoET), which provides images of whole biological cells with high-resolution cellular contrast. Combining these two techniques by performing optical imaging under conditions that do not damage the sample for cryoET allows the combination of the high sensitivity and specificity from single-molecule fluorescence with the cellular context from cryoET. In this work, we use PAmKate, a red photoactivatable fluorescent protein, to perform cryogenic single-molecule imaging of proteins in the model organism Caulobacter crescentus at 77 K with sufficiently low illumination powers to prevent damage of the cryogenic sample. The enhanced number of photons detected allows localization precision to be improved to values below 10 nm.

Nucleoid-associated proteins (NAPs) are DNA-binding proteins critical for the organization and function of the bacterial chromosome. A subclass of NAPs, includingCaulobacter crescentusGapR andEscherichia coliH-NS, preferentially bind AT-rich regions of the nucleoid, but phylogenetic groups that encode GapR rarely encode H-NS. Here, utilizing genetic, biochemical, and biophysical studies of GapR in light of a recent DNA-bound crystal structure of GapR (Guo et al, 2018), we show that although evolutionarily distant, GapR and H-NS possess two regions that are structurally and functionally conserved. These regions are involved in self-association and DNA-binding, even though the two proteins oligomerize and regulate transcription differently. Functional analysis of GapR and H-NS protein chimeras identified structural elements present in H-NS but absent in GapR that rationalize differences in transcriptional regulation. In addition, we identified a sequence element unique to GapR that enables assembly into its tetrameric state. Using fluid-atomic force microscopy, we showed that GapR is capable of bridging DNA moleculesin vitro. Together, these results demonstrate that two distantly related NAPs utilize evolutionarily conserved structural elements to serve specialized cellular roles via distinct mechanisms.

CTCF and cohesin play critical roles in organizing mammalian genomes into topologically associating domains (TADs). Here, by combining genetic engineering with quantitative super-resolution Stimulated Emission Depletion (STED) microscopy, we demonstrate that in living cells CTCF forms clusters typically containing 2–8 molecules. A fraction of CTCF clusters, enriched for those with greater than three molecules, are coupled with cohesin complexes with a characteristic physical distance suggestive of a defined molecular interaction. Acute degradation of the cohesin unloader WAPL or transcriptional inhibition result in increased CTCF clustering. Furthermore, the effect of transcriptional inhibition on CTCF clusters is alleviated by the acute loss of the cohesin subunit SMC3. Our study provides quantitative characterization of CTCF clusters in living cells, uncovers the opposing effects of cohesin and transcription on CTCF clustering, and highlights the power of quantitative super-resolution microscopy as a tool to bridge the gap between biochemical and genomic methodologies in chromatin research.

Expansion segments (ESs) are enigmatic insertions within the eukaryotic ribosome, the longest of which resemble tentacle-like extensions that vary in length and sequence across evolution, with a largely unknown function. By selectively engineering rRNA in yeast, we find that one of the largest ESs, ES27L, has an unexpected function in translation fidelity. Ribosomes harboring a deletion in the distal portion of ES27L have increased amino acid misincorporation, as well as readthrough and frameshifting errors. By employing quantitative mass spectrometry, we further find that ES27L acts as an RNA scaffold to facilitate binding of a conserved enzyme, methionine amino peptidase (MetAP). We show that MetAP unexpectedly controls the accuracy of ribosome decoding, which is coupled to an increase in its enzymatic function through its interaction with ES27L. These findings reveal that variable ESs of the ribosome serve important functional roles and act as platforms for the binding of proteins that modulate translation across evolution.

RNA aptamers that generate a strong fluorescence signal upon binding a nonfluorescent small-molecule dye offer a powerful means for the selective imaging of individual RNA species. Unfortunately, conventional in vitro discovery methods are not efficient at generating such fluorescence-enhancing aptamers, because they primarily exert selective pressure based on target affinity—a characteristic that correlates poorly with fluorescence enhancement. Thus, only a handful of fluorescence-enhancing aptamers have been reported to date. In this work, we describe a method for converting DNA libraries into “gene-linked RNA aptamer particles” (GRAPs) that each display ∼105 copies of a single RNA sequence alongside the DNA that encodes it. We then screen large libraries of GRAPs in a high-throughput manner using the FACS instrument based directly on their fluorescence-enhancing properties. Using this strategy, we demonstrate the capability to generate fluorescence-enhancing aptamers that produce a variety of different...

Optical imaging of whole, living animals has proven to be a powerful tool in multiple areas of preclinical research and has allowed noninvasive monitoring of immune responses, tumor and pathogen growth, and treatment responses in longitudinal studies. However, fluorescence-based studies in animals are challenging because tissue absorbs and autofluoresces strongly in the visible light spectrum. These optical properties drive development and use of fluorescent labels that absorb and emit at longer wavelengths. Here, we present a far-red absorbing fluoromodule–based reporter/probe system and show that this system can be used for imaging in living mice. The probe we developed is a fluorogenic dye called SC1 that is dark in solution but highly fluorescent when bound to its cognate reporter, Mars1. The reporter/probe complex, or fluoromodule, produced peak emission near 730 nm. Mars1 was able to bind a variety of structurally similar probes that differ in color and membrane permeability. We demonstrated that a tool kit of multiple probes can be used to label extracellular and intracellular reporter–tagged receptor pools with 2 colors. Imaging studies may benefit from this far-red excited reporter/probe system, which features tight coupling between probe fluorescence and reporter binding and offers the option of using an expandable family of fluorogenic probes with a single reporter gene.

Ease of genetic encoding, labeling specificity, and high photostability are the most sought after qualities in a fluorophore for biological detection. Furthermore, many applications can gain from the fluorogenic nature of fluoromodules and the ability to turn on the same fluoromodules multiple times. Fluorogen-activating peptides (FAPs) bind noncovalently to their cognate fluorogens and exhibit enhanced photostability. Herein, the photostabilities of malachite green (MG)-binding and thiazole-orange-binding FAPs are compared under limiting- and excess-fluorogen conditions to establish distinct mechanisms for photostability that correspond to the dissociation rate of the FAP-fluorogen complex. FAPs with slow dissociation show evidence of dye encapsulation and protection from photo or environmental degradation and single-step bleaching at the single molecule level, whereas those with rapid dissociation show repeated cycles of binding and enhanced photostability by exchange of bleached fluorogen with a new dye. A combination of generalizable selection pressure based on bleaching, flow cytometry, and site-specific amino acid mutagenesis is used to obtain a modified FAP with enhanced photostability, due to rapid dissociation of the MG fluorogen. These studies shed light on the basic mechanisms by which noncovalent association can effect photostable labeling, and demonstrate novel reagents for photostable and intermittent labeling of biological targets.

While DNA-directed nanotechnology is now a well-established platform for bioinspired nanoscale assembly in vitro, the direct targeting of various nanomaterials in living biological systems remains a significant challenge. Hybrid biological systems with integrated and targeted nanomaterials may have interesting and exploitable properties, so methods for targeting various nanomaterials to precise biological locations are required. Fluorescence imaging has benefited from the use of nanoparticles with superior optical properties compared to fluorescent organic dyes or fluorescent proteins. While single-particle tracking (SPT) in living cells with genetically encoded proteins is limited to very short trajectories, the high photon output of genetically targeted and multiplexed quantum dots (QDs) would enable long-trajectory analysis of multiple proteins. However, challenges with genetic targeting of QDs limit their application in these experiments. In this report, we establish a modular method for targeting QD nanoparticles selectively to multiple genetically encoded tags by precomplexing QD-streptavidin conjugates with cognate biotinylated hapten molecules. This approach enables labeling and SPT of multiple genetically encoded proteins on living cells at high speed and can label expressed proteins in the cytosol upon microinjection into living cells. While we demonstrate labeling with three distinct QD conjugates, the approach can be extended to other specific hapten-affinity molecule interactions and alternative nanoparticles, enabling precise directed targeting of nanoparticles in living biological systems.

The rapid development in fluorescence microscopy and imaging techniques has greatly benefited our understanding of the mechanisms governing cellular processes at the molecular level. In particular, super-resolution microscopy methods overcome the diffraction limit to observe nanoscale cellular structures with unprecedented detail, and single-molecule tracking provides precise dynamic information about the motions of labeled proteins and oligonucleotides. Enhanced photostability of fluorescent labels (i.e., maximum emitted photons before photobleaching) is a critical requirement for achieving the ultimate spatio-temporal resolution with either method. While super-resolution imaging has greatly benefited from highly photostable fluorophores, a shortage of photostable fluorescent labels for bacteria has limited its use in these small but relevant organisms. In this study, we report the use of a highly photostable fluoromodule, dL5, to genetically label proteins in the Gram-negative bacterium Caulobacter crescentus, enabling long time-scale protein tracking and super-resolution microscopy. dL5 imaging relies on the activation of the fluorogen Malachite Green (MG), and can be used to label proteins sparsely, enabling single protein detection in live bacteria without initial bleaching steps. dL5-MG complexes emit two-fold more photons before photobleaching compared to organic dyes such as Cy5 and Alexa 647 in vitro; and five-fold more photons compared to eYFP in vivo. We imaged fusions of dL5 to three different proteins in live Caulobacter cells using stimulated emission depletion (STED) microscopy, yielding a four-fold resolution enhancement compared to diffraction-limited imaging. Importantly, dL5 fusions to an intermediate filament protein CreS are significantly less perturbative compared to traditional fluorescent protein fusions. To the best of our knowledge, this is the first demonstration of the use of fluorogen activating proteins for super-resolution imaging in live bacterial cells.

Nanoscale localization of single molecules is a crucial function in several advanced microscopy techniques, including single-molecule tracking and wide-field super-resolution imaging 1. To date, a central consideration of such techniques is how to optimize the precision of molecular localization. However, as these methods continue to push toward the nanometre size scale, an increasingly important concern is the localization accuracy. In particular, single fluorescent molecules emit with an anisotropic radiation pattern of an oscillating electric dipole, which can cause significant localization biases using common estimators 2-5. Here we present the theory and experimental demonstration of a solution to this problem based on azimuthal filtering in the Fourier plane of the microscope. We do so using a high efficiency dielectric metasurface polarization/phase device composed of nanoposts with sub-wavelength spacing 6. The method is demonstrated both on fluorophores embedded in a polymer matrix, and in dL5 protein complexes that bind Malachite green 7, 8.

The alteration of cellular functions by anchoring macromolecules to specified organelles may reveal a new area of therapeutic potential and clinical treatment. In this work, a unique phenotype was evoked by influencing cellular behavior through the modification of subcellular structures with genetically targetable macromolecules. These fluorogen-functionalized polymers, prepared via controlled radical polymerization, were capable of exclusively decorating actin, cytoplasmic, or nuclear compartments of living cells expressing localized fluorgen-activating proteins. The macromolecular fluorogens were optimized by establishing critical polymer architecture-biophysical property relationships which impacted binding rates, binding affinities, and the level of internalization. Specific labeling of subcellular structures was realized at nanomolar concentrations of polymer, in the absence of membrane permeabilization or transduction domains, and fluorogen-modified polymers were found to bind to protein intact after delivery to the cytosol. Cellular motility was found to be dependent on binding of macromolecular fluorogens to actin structures causing rapid cellular ruffling without migration.

Agonist-promoted G-protein coupled receptor (GPCR) endocytosis and recycling plays an important role in many signaling events in the cell. However, the approaches that allow fast and quantitative analysis of such processes still remain limited. Here we report an improved labeling approach based on the genetic fusion of a fluorogen activating protein (FAP) to a GPCR and binding of a sulfonated analog of the malachite green (MG) fluorogen to rapidly and selectively label cell surface receptors. Fluorescence microscopy and flow cytometry demonstrate that this dye does not cross the plasma membrane, binds with high affinity to a dL5** FAP-GPCR fusion construct, activating tagged surface receptors within seconds of addition. The ability to rapidly and selectively label cell surface receptors with a fluorogenic genetically encoded tag allows quantitative imaging and analysis of highly dynamic processes like receptor endocytosis and recycling.

Laurdan Identifies Different Lipid Membranes in Eukaryotic Cells Enrico Gratton and Michelle A. Digman CONTENTS 13.1 Introduction 283 13.1.1 Spectroscopic Properties of Laurdan 283 13.2 The Phasor Approach to Spectral and Lifetime Analysis 287 13.3 The Lifetime Phasor Transformation and Its Interpretation 289 13.4 Results of the Analysis of the Emission of Laurdan Using Spectral and Lifetime Phasors in GUVs Model Systems 291 13.5 The Lifetime Phasor for Laurdan in GUVs 292 13.6 Live Cell Membrane Fluidity 295 13.6.1 Spectral Phasors 295 13.6.2 Lifetime Phasors in Live 3T3 Cells 297 13.7 Conclusions and Further Considerations 299 13.8 Methods 300 13.8.1 Preparation of the GUVs 300 13.8.2 NIH3T3 Cell Cultures 301 13.8.3 FLIM Analysis 301 13.8.4 Spectral Analysis 301 Acknowledgment 301 References 302 13.1 INTRODUCTION 13.1.1 S pectroscopic P roperties of L aurdan There are several commonly used approaches for the study of membrane properties of live cells based on fluorescence probes. In one approach, lipids with specific fluores- cent markers are incorporated in the cell membranes. The advantage of this approach is that it is possible to study the membrane distribution of specific lipids. However, when the aim of the study is to detect membrane microdomains independently of their

Chemistry

In our paper published in 20131, we reported on the influence of molecular crowding on gene-expression dynamics, specifically promoting the robustness of gene expression. We also established the first multiscale synthetic biology approach (single molecule ex vivo artificial cells) for the study of gene regulation using cells as natural guiding principles. Our paper demonstrates that the impact of molecular crowding on gene expression can be modulated by genetic components, a negative feedback loop and crowding composition.

Studies on diffusion dynamics of single molecules (SMs) have been useful in revealing inhomogeneity of polymer thin films near and above the glass-transition temperature (T-g). However, despite several applications of polymer thin films where exposure to solvent (or vapor) is common, the effect of absorbed solvent molecules on local morphology and rigidity of polymer, matrices is yet to be explored in detail. High-T-g hydrophilic polymers such as poly(vinylpyrrolidone) (PVP) are used as pharmaceutical coatings for drug release in aqueous medium, as they readily absorb moisture, which results in effective lowering. of the T-g and thereby leads to plasticization. The effect of moisture absorption on swelling and softening of PVP thin films was investigated by visualizing the diffusion dynamics of rhodamine 6G (Rh6G) tracer molecules at various ambient relative humidities (RH). Wide-field epifluorescence microscopy, in conjunction with high-resolution SM tracking, was used to monitor the spatiotemporal evolution of individual tracers under varied moisture contents of the matrix. In the absence of atmospheric moisture, Rh6G molecules in dry PVP films are translationally inactive, suggestive of rigid local environments. Under low moisture contents (RH 30-50%), translational mobility remains arrested but rotational motion is augmented, indicating slight swelling of the polymer network which marks the onset of plasticization. The translational mobility of Rh6G was found to be triggered only at a threshold ambient RH, beyond which a large proportion of tracers exhibit extensive diffusion dynamics. Interestingly, SM tracking data at higher moisture contents of the film (RH >= 60%) reveal that the distributions of dynamic parameters (such as diffusivity) are remarkably broad, spanning several orders of magnitude. Furthermore, Rh6G molecules display a wide variety of translational motion even at a fixed ambient RH, clearly pointing out the extremely inhomogeneous environment of plasticized PVP network. Intriguingly, it is observed that a majority of tracers undergo anomalous subdiffusion even under high moisture contents of the matrix. Analyses of SM trajectories using velocity autocorrelation function reveal that subdiffusive behaviors of Rh6G are likely to originate from fractional Brownian motion, a signature of tracer dynamics in viscoelastic medium.

Summary The integration of synthetic and cell-free biology has made tremendous strides towards creating artificial cellular nanosystems using concepts from solution-based chemistry: only the concentrations of reacting species modulate gene expression rates. However, it is known that macromolecular crowding, a key feature of natural cells, can dramatically influence biochemical kinetics by volume exclusion effects that reduce diffusion rates and enhance binding rates of macromolecules. Here, we demonstrate that macromolecular crowding can increase the robustness of gene expression through integrating synthetic cellular components of biological circuits and artificial cellular nanosystems. In addition, we reveal how ubiquitous cellular modules, including genetic components, a negative feedback loop, and the size of crowding molecules, can fine tune gene circuit response to molecular crowding. By bridging a key gap between artificial and living cells, our work has implications for efficient and robust control of both synthetic and natural cellular circuits.

Fluorogen Activating Peptides (FAPs) are protein reporters which activate the fluorescence of a non-covalently bound dark molecule (fluorogen). Light-harvesting dendrimers consisting of multiple chromophores have been built to improve the brightness of a biosensor. The multiple donor dyes transfer energy to the lone acceptor, thus increasing the brightness of the biosensor. Although bright, these light harvesting structures (dyedrons) displayed reduced association constants with the cognate protein, and reduced photostability of the fluorogen-FAP complex. To understand and potentially overcome these limitations, we have synthesized Cy3-Malachite Green based donor-acceptor structures with linker lengths from 10 to 30 atoms and found that longer linker lengths correspond to reduced quantum yield, but not reduced energy transfer efficiency, and that this arises from protein-dye changes that are slow relative to protein-dye association. Ensemble and single molecule level investigation of triplet state quenchers (TSQs) covalently linked to the fluorogen and the tandem dyes provide deeper insights into the development of bright, photostable probes for imaging applications.

The ability to detect single molecules over the electronic noise requires high performance detector systems. Electron Multiplying Charge-Coupled Device (EMCCD) cameras have been employed successfully to image single molecules. Recently, scientific Complementary Metal Oxide Semiconductor (sCMOS) based cameras have been introduced with very low read noise at faster read out rates, smaller pixel sizes and a lower price compared to EMCCD cameras. In this study, we have compared the two technologies using two EMCCD and three sCMOS cameras to detect single Cy5 molecules. Our findings indicate that the sCMOS cameras perform similar to EMCCD cameras for detecting and localizing single Cy5 molecules.

The integration of optofluidics and soft materials has ushered in a new generation of flexible devices for drug delivery, biosensors, and tissue engineering. These devices are biocompatible and allow complex control of device dynamics using fluidic and pneumatic controls. Importantly, these devices could be combined with synthetic biological systems to increase sensory and control capabilities of the devices through exploitation of complex genetic controls in synthetic cells. Here, we take the first step towards such bio-opto-fluidic systems by constructing a hybrid device that consists of soft materials, synthetic bacteria, fluidic systems and electronics. Specifically our device consists of a flexible polydimethylsiloxane (PDMS) chamber for culturing synthetic Escherichia coli that express green fluorescent proteins (GFP) and a flexible electronic layer housing an LED with an emission spectrum peak at 395 nm. The PDMS chamber has high gas permeability that facilitates aerobic cell growth conditions, high translucence that allows for optical control of the synthetic bacteria, and high viscoelasticity that provides mechanical versatility. We demonstrated that the synthetically modified bacteria can be excited internally by an electronic component without sacrificing the flexibility and transparency of the device. We have also shown that isopropyl β-D-1-thiogalactopyranoside (IPTG) can be delivered via micro channels over a flexible micro-porous PDMS membrane to modulate gene expression of the synthetically modified bacteria inside the device. Furthermore we optimized the geometry of the device with respect to bacterial growth rates, fluorescence expression, and fluid flow properties. Finally, we tested the device during mechanical deformation by bending to demonstrate the robust function of the device in strain-induced conditions. Our work will have wide impact on the development of the next-generation bio-opto-fluidic devices and the integration of synthetic biological systems with soft electronic materials.

Inefficiency of fluorescent labels imposes key limitations in fluorescence microscopy. Photobleaching, non-specific signal from multiple fluorophores, and lack of multiplexing and orthogonal labels impose major challenges in studying complex cellular systems, both at ensemble and single molecule levels. We have addressed these problems by exploiting the binding kinetics of fluorogen activating peptides that on binding to a non-fluorescent molecule (a fluorogen) exhibit fluorescence. Novel methods in flow cytometry and rational design of peptides enabled the elucidation of peptides and fluorogen pairs with varying off rates. Complexes with fast off rates (koff∼5E-3 s-1) can exchange a bleached fluorogen molecule with a fresh fluorogen molecule in solution. Since the free fluorogen is dark, we can image for extended time scales in the presence of excess fluorogen. For complexes with slow off rates (koff∼5E-5 s−1) and high affinity (Kd

Biological imaging has profited from the development of a variety of fluorescent probes such as organic dyes, quantum dots and fluorescent proteins. More recently, Fluorogen Activating Peptides (FAP) have been developed through directed evolution. These FAPs bind non-covalently to an otherwise non-fluorescent molecule (fluorogen) to form fluorescent complexes. FAPs have been successfully employed in various live-cell imaging applications in the past two years. For live-cell and single molecule imaging applications, photostability is a key requirement for any fluorescent probe. FAPs have been shown to exhibit high photostability in cell-imaging applications. An understanding of the mechanism underlying the photostability of these FAPs will help us in future to develop FAP clones with higher photostability. In this study, we have characterized the photobleaching behavior of two Malachite green (MG) binding FAP clones: dL5NP138 and HL4. These studies have been performed at a single molecule level. These studies have shown FAPs to be more photostable compared to a widely used single molecule dye, Cy5, at a single molecule condition. Additionally we have exposed FAPs and the Green fluorescent protein to vigorous oxidizing conditions to assess the encapsulation of the chromophore in the protein environment.Our studies show that FAPs protect MG (the fluorogen) from external environments that are rich in reactive oxygen species. The same behavior was observed by GFP compared to a synthetic soluble GFP chromophore. Using single molecule approaches in the presence of excess dye, we have demonstrated that these MG-binding FAPs are photostable through encapsulation of the fluorogen, and not by exchange of the bleached fluorogen. This mechanism provides MG-FAP complexs that can be used for single molecule detection over periods greater than Cy5 labels, yet with the convenience of genetically encoded reporters such as GFP.