Search

Antimicrobial photodynamic therapy (APDT) employs a photosensitizer, light, and molecular oxygen to treat infectious diseases via oxidative damage, with a low likelihood for the development of resistance. For optimal APDT efficacy, photosensitizers with cationic charges that can permeate bacteria cells and bind intracellular targets are desired to not limit oxidative damage to the outer bacterial structure. Here we report the application of brominated DAPI (Br-DAPI), a water-soluble, DNA-binding photosensitizer for the eradication of both Gram-negative and Gram-positive bacteria (as demonstrated on N99 Escherichia coli and Bacillus subtilis, respectively). We observe intracellular uptake of Br-DAPI, ROS-mediated bacterial cell death via one- and two-photon excitation, and selective photocytotoxicity of bacteria over mammalian cells. Photocytotoxicity of both N99 E. coli and B. subtilis occurred at submicromolar concentrations (IC50 = 0.2-0.4 μM) and low light doses (5 min irradiation times, 4.5 J cm-2 dose), making it superior to commonly employed APDT phenothiazinium photosensitizers such as methylene blue. Given its high potency and two-photon excitability, Br-DAPI is a promising novel photosensitizer for in vivo APDT applications.

Human carboxylesterase 2 (hCES2) converts anticancer prodrugs, such as irinotecan, into their active metabolites

A highly flexible, integrated system for preparing samples for serial synchrotron crystallography at room and cryogenic temperatures is described and evaluated. The system is compatible with the existing infrastructure for high-throughput crystallography at synchrotrons and addresses major issues in maximizing data quantity and quality from large numbers of small crystals., Serial synchrotron crystallography (SSX) is enabling the efficient use of small crystals for structure–function studies of biomolecules and for drug discovery. An integrated SSX system has been developed comprising ultralow background-scatter sample holders suitable for room and cryogenic temperature crystallographic data collection, a sample-loading station and a humid ‘gloveless’ glovebox. The sample holders incorporate thin-film supports with a variety of designs optimized for different crystal-loading challenges. These holders facilitate the dispersion of crystals and the removal of excess liquid, can be cooled at extremely high rates, generate little background scatter, allow data collection over >90° of oscillation without obstruction or the risk of generating saturating Bragg peaks, are compatible with existing infrastructure for high-throughput cryocrystallography and are reusable. The sample-loading station allows sample preparation and loading onto the support film, the application of time-varying suction for optimal removal of excess liquid, crystal repositioning and cryoprotection, and the application of sealing films for room-temperature data collection, all in a controlled-humidity environment. The humid glovebox allows microscope observation of the sample-loading station and crystallization trays while maintaining near-saturating humidities that further minimize the risks of sample dehydration and damage, and maximize working times. This integrated system addresses common problems in obtaining properly dispersed, properly hydrated and isomorphous microcrystals for fixed-orientation and oscillation data collection. Its ease of use, flexibility and optimized performance make it attractive not just for SSX but also for single-crystal and few-crystal data collection. Fundamental concepts that are important in achieving desired crystal distributions on a sample holder via time-varying suction-induced liquid flows are also discussed.

Supported lipid bilayers (SLBs) hold tremendous promise as cellular-mimetic structures that can be readily interfaced with analytical and screening tools. The incorporation of transmembrane proteins, a key component in biological membranes, is a significant challenge that has limited the capacity of SLBs to be used for a variety of biotechnological applications. Here, we report an approach using a cell-free expression system for the cotranslational insertion of membrane proteins into hybrid-supported lipid bilayers (HSLBs) containing phospholipids and diblock copolymers. We use cell-free expression techniques and a model transmembrane protein, the large conductance mechanosensitive channel (MscL), to demonstrate two routes to integrate a channel protein into a HSLB. We show that HSLBs can be assembled with integrated membrane proteins by either cotranslational integration of protein into hybrid vesicles, followed by fusion of these proteoliposomes to form a HSLB, or preformation of a HSLB followed by the cell-free synthesis of the protein directly into the HSLB. Both approaches lead to the assembly of HSLBs with oriented proteins. Notably, using single-particle tracking, we find that the presence of diblock copolymers facilitates membrane protein mobility in the HSLBs, a critical feature that has been difficult to achieve in pure lipid SLBs. The approach presented here to integrate membrane proteins directly into preformed HSLBs using cell-free cotranslational insertion is an important step toward enabling many biotechnology applications, including biosensing, drug screening, and material platforms requiring cell membrane-like interfaces that bring together the abiotic and biotic worlds and rely on transmembrane proteins as transduction elements.

Lightsheet microscopy offers an ideal method for imaging of large (mm-cm scale) biological tissues rendered transparent via optical clearing protocols. However the diversity of clearing technologies and tissue types, and how these are adapted to the microscope can make tissue mounting glitchy and somewhat irreproducible. Tissue preparation for imaging can involve glues and or equilibration in a variety of expensive and/or proprietary formulations. Here we present practical advice for mounting and capping cleared tissues in optical cuvettes for macroscopic imaging, providing a standardized 3D cell that can be imaged routinely and relatively inexpensively. We show that acrylic cuvettes should be non-aberrating with objective numerical apertures less than 0.65, and present an inexpensive tool for alignment and calibration of standard lightsheet parameters. Mouse embryo, liver and heart imaging are demonstrated as examples with practical recommendations for acquisition and post-processing.

The composition, stoichiometry and interactions of supramolecular protein complexes are a critical determinant of biological function. Several techniques have been developed to study molecular interactions and quantify subunit stoichiometry at the single molecule level; however, these typically require artificially low expression levels to achieve the low fluorophore concentration required for single molecule imaging, or use of detergent isolation of complexes that may perturb native subunit interactions. Here we present an alternative approach where protein complexes are assembled at physiological concentrations and subsequently diluted in situ for single-molecule level observations while preserving them in a near-native cellular environment. We show that coupling this in situ dilution strategy with single molecule techniques such as in vivo Fluorescence Correlation Spectroscopy (FCS), bleach step counting for quantifying protein complex stoichiometry, and two-color single molecule colocalization, improves the quality of data obtained using these single molecule fluorescence methods. Single Protein Recovery After Dilution (SPReAD) is a simple and versatile means of extending the concentration range of single molecule measurements into the cellular regime while minimizing potential artifacts and perturbations of protein complex stoichiometry.SIGNIFICANCE STATEMENTQuantifying the composition and stoichiometry of protein complexes in live cells is critical to understanding mechanisms involved in their function. Here we detail a method in which protein complexes are assembled intracellularly at physiological concentrations, but then diluted to levels suitable for single-molecule fluorescence observations while still within a cellular environment. The technique permits the use of common single molecule analysis techniques such as stepwise photobleaching quantification and fluorescence correlation spectroscopy to determine stoichiometry and functional interactions while avoiding artifacts that may occur from the use of detergent isolation methods or from the artificially low expression levels sometimes used to attain single molecule observation levels.

Precise pH measurements in the immediate environment of receptors is essential for elucidating the mechanisms through which local pH changes associated with diseased phenotypes manifest into aberrant receptor function. However, current pH sensors lack the ability to localize and target specific receptor molecules required to make these measurements. Herein we present the Litmus-body, our recombinant protein-based pH sensor, which through fusion to an anti-IgG nanobody is capable of piggybacking on IgG antibodies for molecular targeting to specific proteins on the cell surface. By normalizing a pH-dependent green fluorescent protein to a long Stokes shift red fluorophore or fluorescent protein, we readily report pH independent of sensor concentration using a single 488 nm excitation. Our Litmus-body showed excellent responsiveness in solution, with a greater than 50-fold change across the regime of physiological pH. The sensor was further validated for use on live cells and shown to be specific to the protein of interest. In complex with our Litmus-body, cetuximab therapeutic antibody retained its functionality in binding and inhibiting ligand interaction of its target epidermal growth factor receptor (EGFR), triggering receptor-mediated endocytosis that allowed tracking of local pH from the cell surface through the endocytic pathway.

The cancer cell glycocalyx serves as a major line of defense against immune surveillance. However, how specific physical properties of the glycocalyx contribute to immune evasion and how these properties are regulated are not well understood. Here, we uncover how the surface density, glycosylation, and crosslinking of cancer-associated mucins contribute to the nanoscale material thickness of the glycocalyx, and further analyze the effect of the glycocalyx thickness on resistance to effector cell attack. Natural Killer (NK) cell-mediated cytotoxicity exhibits a near perfect inverse correlation with the glycocalyx thickness of target cells regardless of the specific glycan structures present. NK cells expressing a chimeric antigen receptor (CAR) have an enhanced ability to breach the glycocalyx and kill target cells. Equipping the NK cell surface with a mucin-digesting enzyme also improves killing with a performance enhancement that rivals or exceeds CARs in some cases. Together, our results provide new considerations for improving cancer immunotherapies.

Azimuthal beam scanning, also referred to as circle scanning, is an effective way of eliminating coherence artifacts with laser illumination in widefield microscopy. With a static excitation spot, dirt on the optics and internal reflections can produce an uneven excitation field due to interference fringes. These artifacts become more pronounced in TIRF microscopy, where the excitation is confined to an evanescent field that extends a few hundred nanometers above the coverslip. Unwanted intensity patterns that arise from these imperfections vary with path of the excitation beam through the microscope optical train, so by rapidly rotating the beam through its azimuth the uneven illumination is eliminated by averaging over the camera exposure time. In addition to being useful from TIRF microscopy, it is also critical for scanning angle interference microscopy (SAIM), an axial localization technique with nanometer-scale precision that requires similar instrumentation to TIRF microscopy. For robust SAIM localization, laser excitation with a homogeneous profile over a range of polar angles is required. We have applied the circle scanning principle to SAIM, constructing an optimized instrument configuration and open-source hardware, enabling high-precision localization and significantly higher temporal resolution than previous implementations. In this chapter, we detail the design and construction of the SAIM instrument, including the optical configuration, required peripheral devices, and system calibration.

Recent advances in fluorescence microscopy have enabled the visualization of subcellular structures at unprecedented resolution. However, the complexity of state-of-the-art microscopes has increased considerably, often requiring the precise control and synchronization of multiple peripheral devices at high speeds. Drawing inspiration from open-source prototyping systems, like the Arduino, we describe the development of a new control platform that adopts the best features of these systems – affordability, facile programmability, and flexible connectivity – but with the scientific-grade inputs and outputs (I/O) and built-in routines that are necessary to control peripherals in advanced microscopy applications. Notably, our platform includes waveform generators and I/O for point-and azimuthal-scanning of excitation in laser-based applications. As a proof of concept, we show how the integration of waveform generation, multiplexed analog outputs, and native hardware triggers into a single central hub provides a versatile platform for performing fast circle-scanning acquisitions, including ring scanning-angle interference microscopy (SAIM), total internal reflection fluorescence (TIRF and ring TIRF) microscopy, and multiangle TIRF (MA-TIRF). We also demonstrate how the low communication latency of our hardware platform can reduce image intensity and reconstruction artifacts arising from synchronization errors produced by software control. Our complete platform, including hardware design files, firmware, API, software, and all associated source code, will be hosted for community-based development and collaboration.

Significance Gaps in our ability to document local water relations in leaves compromise our ability to build complete models of leaf and plant function and our understanding of ecophysiological phenomena, such as response and adaptation to drought. Macroscopically, leaf water potential has been shown to impact vegetative growth and yield, susceptibility to disease, and, in extreme drought, plant viability, making it a promising candidate trait to improve water-use efficiency in plants. In this paper, we present a nanoscale sensor (AquaDust) that provides minimally disruptive measurements of water potential in leaves of intact plants at high spatial and temporal resolution. This creates opportunities for improving our understanding of the mechanisms coupling variations in water potential to biological and physical processes., Leaf water potential is a critical indicator of plant water status, integrating soil moisture status, plant physiology, and environmental conditions. There are few tools for measuring plant water status (water potential) in situ, presenting a critical barrier for developing appropriate phenotyping (measurement) methods for crop development and modeling efforts aimed at understanding water transport in plants. Here, we present the development of an in situ, minimally disruptive hydrogel nanoreporter (AquaDust) for measuring leaf water potential. The gel matrix responds to changes in water potential in its local environment by swelling; the distance between covalently linked dyes changes with the reconfiguration of the polymer, leading to changes in the emission spectrum via Förster Resonance Energy Transfer (FRET). Upon infiltration into leaves, the nanoparticles localize within the apoplastic space in the mesophyll; they do not enter the cytoplasm or the xylem. We characterize the physical basis for AquaDust’s response and demonstrate its function in intact maize (Zea mays L.) leaves as a reporter of leaf water potential. We use AquaDust to measure gradients of water potential along intact, actively transpiring leaves as a function of water status; the localized nature of the reporters allows us to define a hydraulic model that distinguishes resistances inside and outside the xylem. We also present field measurements with AquaDust through a full diurnal cycle to confirm the robustness of the technique and of our model. We conclude that AquaDust offers potential opportunities for high-throughput field measurements and spatially resolved studies of water relations within plant tissues.

Coordination compounds of the lanthanide ions (Ln(3+)) have important applications in medicine due to their photophysical, magnetic, and nuclear properties. To effectively use the Ln(3+) ions for these applications, chelators that stably bind them in vivo are required to prevent toxic side effects that arise from localization of these ions in off-target tissue. In this study, two new picolinate-containing chelators, a heptadentate ligand OxyMepa and a nonadentate ligand Oxyaapa, were prepared, and their coordination chemistries with Ln(3+) ions were thoroughly investigated to evaluate their suitability for use in medicine. Protonation constants of these chelators and stability constants for their Ln(3+) complexes were evaluated. Both ligands exhibit a thermodynamic preference for small Ln(3+) ions. The log K(LuL) = 12.21 and 21.49 for OxyMepa and Oxyaapa, respectively, indicating that the nonadentate Oxyaapa forms complexes of significantly higher stability than the heptadentate OxyMepa. X-ray crystal structures of the Lu(3+) complexes were obtained, revealing that Oxyaapa saturates the coordination sphere of Lu(3+), whereas OxyMepa leaves an additional open coordination site for a bound water ligand. Solution structural studies carried out with NMR spectroscopy revealed the presence of two possible conformations for these ligands upon Ln(3+) binding. Density functional theory (DFT) calculations were applied to probe the geometries and energies of these conformations. Energy differences obtained by DFT are small, but consistent with experimental data. The photophysical properties of the Eu(3+) and Tb(3+) complexes were characterized, revealing modest photoluminescent quantum yields of < 2%. Luminescence lifetime measurements were carried out in H(2)O and D(2)O, showing that the Eu(3+) and Tb(3+) complexes of OxyMepa have two inner-sphere water ligands, whereas the Eu(3+) and Tb(3+) complexes of Oxyaapa have zero. Lastly, variable-temperature (17)O NMR spectroscopy was performed for the Gd-OxyMepa complex to determine its water exchange rate constant of [Formula: see text] = (2.8 ± 0.1) × 10(6) s(−1). Collectively, this comprehensive characterization of these Ln(3+) chelators provides valuable insight for their potential use in medicine and garners additional understanding on ligand design strategies.

Developing labelling methods that densely and specifically label targeted cellular structures is critically important for centroid localization-based super-resolution microscopy. Being easy and inexpensive to produce in the laboratory and of relatively small size, RNA aptamers have potential as a substitute for conventional antibody labelling. By using aptamers selected against common protein tags - GFP (green fluorescent protein) in this case - we demonstrate labelling methods using dSTORM-compatible fluorophores for STORM and hybridizable imager strands for DNA-PAINT super-resolution optical imaging of any cellular proteins fused to the aptamer binding target. We show that we can label both extracellular and intracellular proteins for super-resolution imaging, and that the method in particular, offers some interesting advantages for live cell super-resolution imaging of plasma membrane proteins. KEY POINTSO_LIA simple to use RNA aptamer method for super-resolution STORM and PAINT imaging in cells expressing common protein tags. C_LIO_LIThe method has a number of unique advantages for live cell imaging at the nanometer scale. C_LIO_LIProvides a means to quantify the number of proteins being trafficked on the plasma membrane. C_LI

Mapping the complex biogeography of microbial communities in situ with high taxonomic and spatial resolution poses a major challenge because of the high density and rich diversity of species in environmental microbiomes and the limitations of optical imaging technology. Here, we introduce High Phylogenetic Resolution microbiome mapping by Fluorescence In-Situ Hybridization (HiPR-FISH), a versatile and cost-effective technology that uses binary encoding and spectral imaging and machine learning based decoding to create micron-scale maps of the locations and identities of hundreds of microbial species in complex communities. We demonstrate the ability of 10-bit HiPR-FISH to distinguish 1023 E. coli strains, each fluorescently labeled with a unique binary barcode. HiPR-FISH, in conjunction with custom algorithms for automated probe design and segmentation of single-cells in the native context of tissues, reveals the intricate spatial architectures formed by bacteria in the human oral plaque microbiome and disruption of spatial networks in the mouse gut microbiome in response to antibiotic treatment. HiPR-FISH provides a framework for analyzing the spatial organization of microbial communities in tissues and the environment at single cell resolution.

Rapid growth and perivascular invasion are hallmarks of glioblastoma (GBM) that have been attributed to the presence of cancer stem-like cells (CSCs) and their association with the perivascular niche. However, the mechanisms by which the perivascular niche regulates GBM invasion and CSCs remain poorly understood due in part to a lack of relevant model systems. To simulate perivascular niche conditions and analyze consequential changes of GBM growth and invasion, patient-derived GBM spheroids were co-cultured with brain endothelial cells (ECs) in microfabricated collagen gels. Integrating these systems with 3D imaging and biochemical assays revealed that ECs increase GBM invasiveness and growth through interleukin-8 (IL-8)-mediated enrichment of CSCs. Blockade of IL-8 inhibited these effects in GBM-EC co-cultures, while IL-8 supplementation increased CSC-mediated growth and invasion in GBM-monocultures. Experiments in mice confirmed that ECs and IL-8 stimulate intracranial tumor growth and invasion in vivo. Collectively, perivascular niche conditions promote GBM growth and invasion by increasing CSC frequency, and IL-8 may be explored clinically to inhibit these interactions.

Femto-Seq is a new nonlinear optical methodology that provides a means to determine the chromatin environment at the base-pair level at and around any site in the nucleus that can be identified by imaging.

Rupture of the ovarian follicle releases the oocyte at ovulation, a timed event that is critical for fertilization. It is not understood how the protease activity required for rupture is directed with precise timing and localization to the outer surface, or apex, of the follicle. We hypothesized that vasoconstriction at the apex is essential for rupture. The diameter and blood flow of individual vessels and the thickness of the apical follicle wall were examined over time to expected ovulation using intravital multiphoton microscopy. Vasoconstriction of apical vessels occurred within hours preceding follicle rupture in wild-type mice, but vasoconstriction and rupture were absent in Amhr2(cre/+)SmoM2 mice in which follicle vessels lack the normal association with vascular smooth muscle. Vasoconstriction is not simply a response to reduced thickness of the follicle wall; vasoconstriction persisted in wild-type mice when thinning of the follicle wall was prevented by infusion of protease inhibitors into the ovarian bursa. Ovulation was inhibited by preventing the periovulatory rise in the expression of the vasoconstrictor endothelin 2 by follicle cells of wild-type mice. In these mice, infusion of vasoconstrictors (either endothelin 2 or angiotensin 2) into the bursa restored the vasoconstriction of apical vessels and ovulation. Additionally, infusion of endothelin receptor antagonists into the bursa of wild-type mice prevented vasoconstriction and follicle rupture. Processing tissue to allow imaging at increased depth through the follicle and transabdominal ultrasonography in vivo showed that decreased blood flow is restricted to the apex. These results demonstrate that vasoconstriction at the apex of the follicle is essential for ovulation.

Fluorescence microscopy methods have seen an increase in popularity in recent years for detecting protein crystals in screening trays. The fluorescence-based crystal detection methods have thus far relied on intrinsic UV-inducible tryptophan fluorescence, nonlinear optics or fluorescence in the visible light range dependent on crystals soaked with fluorescent dyes. In this paper data are presented on a novel visible-light-inducible autofluorescence arising from protein crystals as a result of general stabilization of conjugated double-bond systems and increased charge delocalization due to crystal packing. The visible-light-inducible autofluorescence serves as a complementary method to bright-field microscopy in beamline applications where accurate crystal centering about the rotation axis is essential. Owing to temperature-dependent chromophore stabilization, protein crystals exhibit tenfold higher fluorescence intensity at cryogenic temperatures, making the method ideal for experiments where crystals are cooled to 100 K with a cryostream. In addition to the non-damaging excitation wavelength and low laser power required for imaging, the method can also serve a useful role for differentiating protein crystals from salt crystals in screening trays.

Despite its relevance in numerous natural and industrial processes, the solubility of molecular oxygen has never been directly measured in capillary condensed liquid water. In this article, we measure oxygen solubility in liquid water trapped within nanoporous samples, in metastable equilibrium with a subsaturated vapor. We show that solubility increases two-fold at moderate subsaturations (RH ~ 0.55). This evolution with relative humidity is in good agreement with a simple thermodynamic prediction using properties of bulk water, previously verified experimentally at positive pressure. Our measurement thus verifies the validity of this macroscopic thermodynamic theory to strong confinement and large negative pressures, where ignificant non-idealities are expected. This effect has strong implications for important oxygen-dependent chemistries in natural and technological contexts., Accepted for publication in Langmuir

Fifteen water-soluble rhenium compounds of the general formula [Re(CO)(3)(NN)(PR(3))](+), where NN is a diimine ligand and PR(3) is either 1,3,5-triaza-7-phosphaadamantane (PTA), tris(hydroxymethyl)phosphine (THP), or 1,4-diacetyl-1,3,7-triaza-5-phosphabicylco[3.3.1]nonane (DAPTA), were synthesized and characterized by multinuclear NMR spectroscopy, IR spectroscopy, and X-ray crystallography. The complexes bearing the THP and DAPTA ligands exhibit triplet-based luminescence in air-equilibrated aqueous solutions with quantum yields ranging from 3.4 to 11.5%. Furthermore, the THP and DAPTA complexes undergo photosubstitution of a CO ligand when irradiated with 365 nm light with quantum yields ranging from 1.1 to 5.5% and sensitize the formation of (1)O(2) with quantum yields as high as 70%. By contrast, all of the complexes bearing the PTA ligand are non-emissive and do not undergo photosubstitution when irradiated with 365 nm light. These compounds were evaluated as photoactivated anticancer agents in human cervical (HeLa), ovarian (A2780), and cisplatin-resistant ovarian (A2780CP70) cancer cell lines. All of the complexes bearing THP and DAPTA exhibited a cytotoxic response upon irradiation with minimal toxicity in the absence of light. Notably, the complex with DAPTA and 1,10-phenanthroline gave rise to an IC(50) value of 6 μM in HeLa cells upon irradiation, rendering it the most phototoxic compound in this library. The nature of the photoinduced cytotoxicity of this compound was explored in further detail. These data indicate that the phototoxic response may result from the release of both CO and the rhenium-containing photoproduct, as well as the production of (1)O(2).

The mechanism of the recently reported photocontrolled cationic polymerization of vinyl ethers was investigated using a variety of catalysts and chain-transfer agents (CTAs) as well as diverse spectroscopic and electrochemical analytical techniques. Our study revealed a complex activation step characterized by one-electron oxidation of the CTA. This oxidation is followed by mesolytic cleavage of the resulting radical cation species, which leads to the generation of a reactive cation–this species initiates the polymerization of the vinyl ether monomer–and a dithiocarbamate radical that is likely in equilibrium with the corresponding thiuram disulfide dimer. Reversible addition–fragmentation type degenerative chain transfer contributes to the narrow dispersities and control over chain growth observed under these conditions. Finally, the deactivation step is contingent upon the oxidation of the reduced photocatalyst by the dithiocarbamate radical concomitant with the production of a dithiocarbamate anion that caps the polymer chain end. The fine-tuning of the electronic properties and redox potentials of the photocatalyst in both the excited and the ground states is necessary to obtain a photocontrolled system rather than simply a photoinitiated system. The elucidation of the elementary steps of this process will aid the design of new catalytic systems and their real-world applications.

SummaryObjectiveCartilage injury can lead to post-traumatic osteoarthritis (PTOA). Immediate post-trauma cellular and structural changes are not widely understood. Furthermore, current cellular-resolution cartilage imaging techniques require sectioning of cartilage and/or use of dyes not suitable for patient imaging. In this study, we used multiphoton microscopy (MPM) data with FDA-approved sodium fluorescein to identify and evaluate the pattern of chondrocyte death after traumatic injury.MethodMature equine distal metacarpal or metatarsal osteochondral blocks (OCBs) were injured by 30 MPa compressive loading delivered over 1 s. Injured and control sites were imaged unfixed and in situ 1 h post-injury with sodium fluorescein using rasterized z-scanning. MPM data was quantified in MATLAB, reconstructed in 3-D, and projected in 2-D to determine the damage pattern.ResultsMPM images (600 per sample) were reconstructed and analyzed for cell death. The overall distribution of cell death appeared to cluster into circular (n = 7) or elliptical (n = 4) patterns (p = 0.006). Dead cells were prevalent near cracks in the matrix, with only 26.3% (SE = 5.0%, p

The kinetics with which promoter-proximal paused RNA polymerase II (Pol II) undergoes premature termination versus productive elongation is central to understanding underlying mechanisms of metazoan transcription regulation. To assess the fate of Pol II quantitatively, we tracked photoactivatable GFP-tagged Pol II at uninduced Hsp70 on polytene chromosomes and showed that Pol II is stably paused with a half-life of 5 min. Biochemical analysis of short nascent RNA from Hsp70 reveals that this half-life is determined by two comparable rates of productive elongation and premature termination of paused Pol II. Importantly, heat shock dramatically increases elongating Pol II without decreasing termination, indicating that regulation acts at the step of paused Pol II entry to productive elongation.

Atlastin, a member of the dynamin superfamily, is known to catalyse homotypic membrane fusion in the smooth endoplasmic reticulum (ER). Recent studies of atlastin have elucidated key features about its structure and function; however, several mechanistic details, including the catalytic mechanism and GTP hydrolysis-driven conformational changes, are yet to be determined. Here, we present the crystal structures of atlastin-1 bound to GDP·AlF(4)(-) and GppNHp, uncovering an intramolecular arginine finger that stimulates GTP hydrolysis when correctly oriented through rearrangements within the G domain. Utilizing Förster Resonance Energy Transfer, we describe nucleotide binding and hydrolysis-driven conformational changes in atlastin and their sequence. Furthermore, we discovered a nucleotide exchange mechanism that is intrinsic to atlastin's N-terminal domains. Our results indicate that the cytoplasmic domain of atlastin acts as a tether and homotypic interactions are timed by GTP binding and hydrolysis. Perturbation of these mechanisms may be implicated in a group of atlastin-associated hereditary neurodegenerative diseases.

Confocal microscopy, a technique that has been extensively applied in cellular biological studies, may also be applied to the visualization and three-dimensional imaging of protein crystals at high resolution on synchrotron beamlines. Protein crystal samples are examined using a commercially available confocal microscope adapted for cryogenic use. A preliminary test using a custom confocal design adapted for beamline use is also presented. The confocal optics configuration is compatible with nonlinear imaging techniques such as two-photon excited fluorescence imaging and second harmonic generation. The possibilities of this method are explored using two modes: fluorescence and reflection confocal. In fluorescence mode, small amounts of dye are introduced into the crystal through soaking or growth conditions. Under such conditions, protein crystals are easily resolved from salts and amorphous precipitates, which do not generally take up dye. Reflection mode, which does not require dye, still exhibits greater resolution and sensitivity to surface detail than conventional wide-field microscopy as a result of the confocal optics configuration. The inherent three-dimensional nature of the method means that on-axis sample views (along the direction of the X-ray beam) can be reconstructed from an off-axis configuration, simplifying the beamline setup and providing uniquely detailed views of cryogenically cooled crystals.

Context.—Multiphoton microscopy (MPM) is a nonlinear imaging approach, providing cellular and subcellular details from fresh (unprocessed) tissue by exciting intrinsic tissue emissions. With miniaturization and substantially decreased cost on the horizon, MPM is an emerging imaging technique with many potential clinical applications.Objectives.—To assess the imaging ability and diagnostic accuracy of MPM for human bladder biopsies.Design.—Seventy-seven fresh bladder biopsies were imaged by MPM and subsequently submitted for routine surgical pathology diagnosis. Twelve cases were excluded because of extensive cautery artifact that prohibited definitive diagnosis. Comparison was made between MPM imaging and gold standard sections for each specimen stained with hematoxylin-eosin.Results.—In 57 of 65 cases (88%), accurate MPM diagnoses (benign or neoplastic) were given based on the architecture and/or the cytologic grade. The sensitivity and specificity of MPM in our study were 90.4% and 76.9%, respectively. A positive (neoplastic) diagnosis on MPM had a high predictive value (94%), and negative (benign) diagnoses were sustained on histopathology in two-thirds of cases. Architecture (papillary versus flat) was correctly determined in 56 of 65 cases (86%), and cytologic grade (benign/low grade versus high grade) was assigned correctly in 38 of 56 cases (68%).Conclusions.—The MPM images alone provided sufficient detail to classify most lesions as either benign or neoplastic using the same basic diagnostic criteria as histopathology (architecture and cytologic grade). Future developments in MPM technology may provide urologists and pathologists with additional screening and diagnostic tools for early detection of bladder cancer. Additional applications of such emerging technologies warrant exploration.

We present the development and characterization of nanoparticles loaded with a custom phosphor; we exploit these nanoparticles to perform quantitative measurements of the concentration of oxygen within three-dimensional (3-D) tissue cultures in vitro and blood vessels in vivo. We synthesized a customized ruthenium (Ru)-phosphor and incorporated it into polymeric nanoparticles via self-assembly. We demonstrate that the encapsulated phosphor is non-toxic with and without illumination. We evaluated two distinct modes of employing the phosphorescent nanoparticles for the measurement of concentrations of oxygen: 1) in vitro, in a 3-D microfluidic tumor model via ratiometric measurements of intensity with an oxygen-insensitive fluorophore as a reference, and 2) in vivo, in mouse vasculature using measurements of phosphorescence lifetime. With both methods, we demonstrated micrometer-scale resolution and absolute calibration to the dissolved oxygen concentration. Based on the ease and customizability of the synthesis of the nanoparticles and the flexibility of their application, these oxygen-sensing polymeric nanoparticles will find a natural home in a range of biological applications, benefiting studies of physiological as well as pathological processes in which oxygen availability and concentration play a critical role.

Quantifying the effects of mechanical loading on the metabolic response of chondrocytes is difficult due to complicated structure of cartilage ECM and the coupled nature of the mechanical stimuli presented to the cells. In this study we describe the effects of fluid flow, particularly hydrostatic pressure and wall shear stress, on the Ca2+ signaling response of bovine articular chondrocytes in 3D culture. Using well-established alginate hydrogel system to maintain spherical chondrocyte morphology, we altered solid volume fraction to change scaffold mechanics. Fluid velocities in the bulk of the scaffolds were directly measured via an optical technique and scaffold permeability and aggregate modulus was characterized to quantify the mechanical stimuli presented to cells. Ca2+ signaling response to direct perfusion of chondrocyte-seeded scaffolds increased monotonically with flow rate and was found more directly dependent on fluid velocity rather than shear stress or hydrostatic pressure. Chondrocytes in alginate scaffolds responded to fluid flow at velocities and shear stresses 2–3 orders of magnitude lower than seen in previous monolayer studies. Our data suggest that flow-induced Ca2+ signaling response of chondrocytes in alginate culture may be due to mechanical signaling pathways, which is influenced by the 3D nature of cell shape. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:793–799, 2012

Objective To test whether multiphoton microscopy (MPM) might allow identification of prostatic and periprostatic structures with magnification and resolution similar to gold standard histopathology. Material and Methods The present study included 95 robotic radical prostatectomy patients who consented to participate in an Institutional Review Board-approved study starting in 2007. The types of specimens used for imaging were excised surgical margins and biopsies, and sections obtained from the excised prostate. The specimens were imaged with a custom-built MPM system. All images were compared with haematoxylin/eosin histopathology of the same specimen. Results MPM of freshly excised, unprocessed and unstained tissue can identify all relevant prostatic and periprostatic structures, such as nerves, blood vessels, capsule, underlying acini and also pathological changes, including prostate cancer. Histological confirmation and correlation of these structures and pathologies have validated the findings of MPM. Conclusions MPM shows great promise as a tool for real-time intra-surgical histopathology without needing excision or administration of contrast agents. The results will, however, need to be confirmed in true surgical settings using a miniaturized MPM microendoscope. Keywords: robotic prostatectomy, multiphoton microscopy, prostate cancer, nerve sparing, histopathology, real-time imaging Introduction Prostate cancer is the most common male cancer, claiming almost 27 000 lives annually [1]. When confined to the gland, it can be successfully treated by surgical removal of the organ, a procedure termed radical prostatectomy, which is performed in 70 000 patients in the US every year. The success of this surgery is judged by three measures: complete removal of the cancer-harbouring glands; preservation of nerves that control sexual function; and preservation of sphincteric structures, which maintain urinary control. Anatomically, the prostate gland is surrounded by a fibrous capsule, with a thickness that varies substantially between different parts of the gland. This incomplete capsule is surrounded, in turn, by a multi-layered, anatomically complex fascia that contains fat cells intermingled with loose areolar tissue, autonomic ganglia, a neural plexus, nerve trunks, arteries and veins. This fascia adheres to the capsule and is pierced by vessels and nerves. Both the erectile nerve, which has a variable branching pattern, and the sphincteric sling, are intermingled with the fascial fat, connective tissue and blood vessels [2]. Cancer cells sometimes migrate beyond the gland and either involve the surrounding nerves or grow into the sphincter – a phenomenon termed extra-prostatic extension (EPE). Since these nerves and any clusters of cancer cells are too small to be visualized by eye or using the ×10–12 magnification of the stereoscope of a surgical robot, the competing goals of cancer extirpation versus preservation of potency and continence have to be balanced during surgery [2]. Surgeons make judgments based on information that includes pre-surgical PSA levels, MRI results, DRE results and intra-surgical subjective cues such as colour changes, fibrosis and ease of separating the different fascial layers. One option for more detailed feedback, intraoperative frozen sections, takes time, could damage the delicate structures one is trying to save, and suffers from sampling errors. As a result, the current methods for discriminating between EPE and the tissues that need to be preserved during surgery are imprecise and have an impact on the outcomes of this surgery. Indeed, this inability to identify cancerous cells and their association with nerves can result in incomplete removal of cancer, resulting in positive surgical margins (10–40% occurrence) [3–6], postoperative impotence due to damage to, or excision of, these nerves (25–70%) [7–11], or both positive margins and an impotent patient. It has been estimated that half the patients who undergo radical prostatectomy require postoperative treatment for erectile dysfunction. In addition to causing psychological and quality-of-life compromises, these complications exact a staggering financial cost on the US health care system. At a rate of US $10 000–20 000 per patient per year spent on doctors' office visits, oral medications, vacuum pumps, intra-cavernous injections and secondary surgeries, this costs the US health care system $350–700 million every year. In addition, certain patients are excluded as candidates for nerve-sparing surgery based on the less-than-optimal preoperative assessments, and thus could be denied a chance for a better postoperative quality of life. In summary, access to high-resolution, high-contrast live imaging of the prostatic capsule, apex, sphincter and the surrounding nerves and tissues would improve surgical decision-making and patient outcomes, and reduce the emotional and financial impact of post-surgical complications. One potential solution is a novel non-linear optical imaging technology: multiphoton microscopy (MPM) [12–14]. MPM relies on the simultaneous absorption of two (or three) low-energy (near-infrared) photons to cause a non-linear excitation, which greatly reduces the potential for cellular damage. Excitation only occurs where there is sufficient photon density (at the point of laser focus), providing intrinsic optical sectioning with a resolution equivalent to traditional confocal microscopy. Tissue penetration is greater than standard confocal microscopy because absorption and scattering are greatly reduced at near-infrared wavelengths compared with the visible or ultraviolet spectrum. Most importantly, by utilizing two-photon excitation in the 700–800 nm range, MPM enables the imaging of fresh, living, unprocessed and unstained tissue by utilizing intrinsic tissue emissions (ITEs). The ITE signal is composed of two components: tissue autofluorescence, in part from nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) in cells, elastin in the connective tissue, and lipofuscin in fat and other cells; and second harmonic generation (SHG), which arises from non-centrosymmetric structures such as tissue collagen [12–14]. We and others have shown that MPM/ITE imaging is capable of generating distinctive optical signals that enable imaging of animal [13] and human [15–17] tissues at sub-micron resolution in three dimensions to a depth of up to 0.5 mm; at image acquisition rates of ≈ one image/second, with >105 pixels per image. While the miniaturization of MPM for integration with robotic surgical equipment is currently in progress, we have carried out a prospective study to assess the feasibility of using MPM/ITE for structure identification in excised prostatic and periprostatic tissue. In all cases, the MPM findings were confirmed by comparison with gold standard haematoxylin/eosin (H&E)-stained histopathology. Specimens were obtained from consenting subjects undergoing robotic radical prostatectomy at Weill Cornell Medical College with Dr Tewari, in a protocol approved by the Institutional Review Board. Samples included periprostatic tissue, specifically surgical margins containing the lateral pelvic fascia (LPF), as well as the prostatic acini, in the form of either biopsies or sections of the gland. In LPF specimens, we confirm our ability to identify its components, such as nerves, arteries, fat and connective tissue, as well as sites of local inflammation. These structures are the ones that must be identified during surgery. In the excised specimens (biopsies or sections), we identify the prostatic acini, the surrounding stroma, and the prostatic capsule. Architectural changes in malignant transformation are used during histopathology to determine tumour grade (Gleason grade), and hence the aggressiveness, of the cancer. We show here that MPM/ITE can provide such architectural information.

Many multiphoton imaging applications would benefit from a larger field of view; however, large field of views (>mm) require low magnification objectives which have low light collection efficiencies. We demonstrate a light collection system mounted on a low magnification objective that increases fluorescence collection by as much as 20-fold in scattering tissues. This peripheral detector results in an effective numerical aperture of collection >0.8 with a 3-4 mm field of view.

Translational mobility is involved in every process in neurobiology—released neurotransmitter diffuses through the synaptic cleft in search of receptor targets, membrane receptors traffic to synaptic sites in the neuron, RNA and other cargo are transported to distal dendrites, and cell signaling

Rapid and direct imaging of microscopic tissue morphology and pathology can be achieved by multiphoton imaging of intrinsic tissue fluorophores and second harmonic signals. Engineering parameters for developing this technology for clinical applications include excitation levels and collection efficiencies required to obtain diagnostic quality images from different tissue types and whether these levels are mutagenic. Here we provide data on typical average powers required for high signal-to-noise in vivo tissue imaging and assess the risk potential of these irradiance levels using a mammalian cell gene mutation assay. Exposure times of ~16 milliseconds per cell to 760 nm, ~200 fs raster-scanned laser irradiation delivered through a 0.75 NA objective produced negligible mutagenicity at powers up to about 50 mW.

Many cartilaginous tissues such as intervertebral disc (IVD) display a heterogeneous collagen microstructure that results in mechanical anisotropy. These structures are responsible for mechanical function of the tissue and regulate cellular interactions and metabolic responses of cells embedded within these tissues. Using collagen gels seeded with ovine annulus fibrosus cells, constructs of varying structure and heterogeneity were created to mimic the circumferential alignment of the IVD. Alignment was induced within gels by contracting annular gels around an inner boundary using both a polyethylene center and alginate center to create a composite engineered IVD. Collagen alignment and heterogeneity were measured using second harmonic generation microscopy. Decreasing initial collagen density from 2.5 mg/mL to 1 mg/mL produced greater contraction of constructs, resulting in gels that were 55% and 6.2% of the original area after culture, respectively. As a result, more alignment occurred in annular-shaped 1 mg/mL gels compared with 2.5 mg/mL gels (p

Background Biologics containing growth factors are frequently used to enhance healing after musculoskeletal injuries. One mechanism of action is thought to be though the ability of biologics to induce homing and migration of endogenous mesenchymal stromal cells (MSCs) to a target tissue. However, the ability of biologics to stimulate chemotaxis (directed migration of cells) and chemokinesis (increase rate of cell migration) of MSCs is unknown. Hypothesis/Purpose The aim of this study was to directly compare the ability of biologics including platelet rich plasma (PRP) and bone marrow concentrate (BMC) to induce MSC migration. The hypothesis was that leukocyte-low platelet rich plasma (Llo PRP) would induce migration to a greater extent than leukocyte-high platelet rich plasma (Lhi PRP) or BMC. Methods Bone marrow-derived MSCs were isolated from 8 horses. Migration of MSCs toward a biologic (BMC, Llo PRP, and Lhi PRP) or the positive control platelet derived growth factor (PDGF) was continuously traced and measured for 24hrs using time-lapse microscopy and a microfluidics device. Cell migration, chemotaxis and chemokinesis were determined by measurements of displacement, number of cells migrated, and cell flux. Results All biologics resulted in a significantly greater percentage of MSCs migrated compared to the positive control (PDGF). MSCs migrated further toward BMC compared to Llo PRP. Cell migration, measured as cell flux, was greater toward BMC and Lhi PRP than Llo PRP. Conclusion The biologics BMC and Lhi PRP elicit greater chemotaxis and chemokinesis of MSCs than Llo PRP. However, all biologics recruited the same number of MSCs suggesting that differences in other regenerative effects, such as growth factor concentration, between biologics should be strongly considered when choosing a biologic for treatment of musculoskeletal injuries. The results of this study have the potential to reduce the need, risks, and costs associated with MSC culture and delivery.

Non-ionic photoacid generators (PAGs) have been designed and synthesized for use in two-photon lithography (TPL). The chromophores in these new PAGs are covalently linked to the photocleavable group by a flexible joint. Their thermal stability, solubility and efficiency to produce acid under both one- and two-photon excitation were characterized. The potential of these PAGs for TPL was tested in two negative-tone resist systems relying on different mechanisms: free-radical/cationic polymerization or a cationically initiated cross-linking reaction. These PAGs needed lower threshold power for polymerization compared to a commercially available photoinitiator, isopropylthioxanthone, and a photoacid generator, N-hydroxynaphthalimide triflate. Microstructures with a resolution of 0.6 µm were fabricated and the threshold power for polymerization was found to be below 2 mW.

Ribbon is a nuclear Broad Tramtrack Bric-a-brac (BTB) -domain protein required for morphogenesis of the salivary gland and trachea. We recently showed that ribbon mutants exhibit decreased Crumbs and Rab11-coincident apical vesicles and increased apical Moesin activity and microvillar structure during tube elongation. To learn how these molecular and morphological changes affect the dynamics of tubulogenesis, we optimized an advanced two-photon microscope to enable high-resolution live imaging of the salivary gland and trachea. Live imaging revealed that ribbon mutant tissues exhibit slowed and incomplete lumenal morphogenesis, consistent with previously described apical defects. Because Moesin activity correlates with cortical stiffness, we hypothesize that ribbon mutants suffer from increased apical stiffness during morphogenesis. We develop this hypothesis through mechanical analysis, using the advantages of live imaging to construct computational elastic and analytical viscoelastic models of tube elongation, which suggest that ribbon mutant tubes exhibit three- to fivefold increased apical stiffness and twofold increased effective apical viscosity. Developmental Dynamics 237:2874–2888, 2008. © 2008 Wiley-Liss, Inc.

Background & Aims: The ability to observe cellular and subcellular detail during routine endoscopy is a major goal in the development of new endoscopic imaging techniques. Multiphoton microscopy, which relies on nonlinear infrared optical processes, has the potential to identify cellular details by excitation of endogenous fluorescent molecules. We examined the feasibility of using multiphoton microscopy to characterize mucosal histology in the human gastrointestinal tract. Methods: A multiphoton microscope was used to determine the optimal excitation wavelength for examination of gastrointestinal mucosa. Fresh, unfixed, and unstained biopsy specimens obtained during routine endoscopy in human subjects then were examined by confocal microscopy and multiphoton microscopy. Multiphoton images also were compared with standard H&E images obtained from paired biopsy specimens. A prototype miniaturized multiphoton probe was used to examine intact rat colon. Results: Peak multiphoton autofluorescence intensity was detected in mucosa excited at 735 nm. Multiphoton microscopic examination of unstained biopsy specimens revealed improved cellular detail relative to either unstained or stained specimens examined by confocal imaging. Resolution of structures such as epithelial nuclei, goblet cells, and interstitial fibers and cells was comparable with what was obtained using standard H&E histology. Similar findings were observed when using a prototype miniaturized multiphoton probe. Conclusions: Multiphoton microscopy can be used to examine gastrointestinal mucosa at the cellular level, without the need for fluorescent dyes. The construction of a multiphoton endomicroscope therefore could provide a practical means of performing virtual biopsies during the course of routine endoscopy, with advantages over currently available endomicroscopy technologies.

Modulation of synaptic transmission is a major mechanism for the functional reconfiguration of neuronal circuits. Neurotransmitter release and, consequently, synaptic strength are regulated by intracellular Ca2+ levels in presynaptic terminals. In identified neurons of the lobster pyloric network, we studied localized, voltage-induced Ca2+ accumulation and its modulation in varicosities on distal neuritic arborizations, which have previously been shown to be sites of synaptic contacts. We previously demonstrated that dopamine (DA) weakens synaptic output from the pyloric dilator (PD) neuron and strengthens synaptic output from the lateral pyloric (LP) and pyloric constrictor (PY) neurons. Here we show that DA modifies voltage-activated Ca2+ accumulation in many varicosities in ways that are consistent with DA's effects on synaptic transmission: DA elevates Ca2+ accumulation in LP and PY varicosities and reduces Ca2+ accumulation in PD varicosities. However, in all three neuron types, we also found varicosities that were unaffected by DA. In the PY neurons, we found that DA can simultaneously increase and decrease voltage-evoked Ca2+ accumulation at different varicosities, even within the same neuron. These results suggest that regulation of Ca2+ entry is a common mechanism to regulate synaptic strength in the pyloric network. However, voltage-evoked local Ca2+ accumulation can be differentially modulated to control Ca2+-dependent processes in functionally separate varicosities of a single neuron.

Bone elongation originates from cartilaginous discs (growth plates) at both ends of a growing bone. Here chondrocytes proliferate and subsequently enlarge (hypertrophy), laying down a matrix that serves as the scaffolding for subsequent bone matrix deposition. Because cartilage is generally avascular, all nutrients, oxygen, signaling molecules, and waste must be transported relatively long distances through the tissue for it to survive and function. Here we examine the transport properties of growth plate cartilage. Ex vivo, fluorescence photobleaching recovery methods are used in tissue explants. In vivo, multiphoton microscopy is used to image through an intact perichondrium and into the cartilage of anesthetized mice. Systemically introduced fluorescent tracers are monitored directly as they move from the vasculature into the cartilage. We demonstrate the existence of a relatively permissive region at the midplane of the growth plate, where chondrocytes transition from late proliferative to early hypertrophic stages and where paracrine communication is known to occur between chondrocytes and cells in the surrounding perichondrium. Transport in the living mouse is also significantly affected by fluid flow from the two chondro-osseus junctions, presumably resulting from a pressure difference between the bone vasculature and the cartilage.

The mechanisms controlling cortical dendrite initiation and targeting are poorly understood. Multiphoton imaging of developing mouse cortex reveals that apical dendrites emerge by direct transformation of the neuron9s leading process during the terminal phase of neuronal migration. During this ∼110 min period, the dendritic arbor increases ∼2.5-fold in size and migration arrest occurs below the first stable branch point in the developing arbor. This dendritic outgrowth is triggered at the time of leading process contact with the marginal zone (MZ) and occurs primarily by neurite extension into the extracellular matrix of the MZ. In reeler cortices that lack the secreted glycoprotein Reelin, a subset of neurons completed migration but then retracted and reorganized their arbor in a tangential direction away from the MZ soon after migration arrest. For these reeler neurons, the tangential oriented primary neurites were longer lived than the radially oriented primary neurites, whereas the opposite was true of wild-type (WT) neurons. Application of Reelin protein to reeler cortices destabilized tangential neurites while stabilizing radial neurites and stimulating dendritic growth in the MZ. Therefore, Reelin functions as part of a polarity signaling system that links dendritogenesis in the MZ with cellular positioning and cortical lamination. SIGNIFICANCE STATEMENT Whether the apical dendrite emerges by transformation of the leading process of the migrating neuron or emerges de novo after migration is completed is unclear. Similarly, it is not clear whether the secreted glycoprotein Reelin controls migration and dendritic growth as related or separate processes. Here, multiphoton microscopy reveals the direct transformation of the leading process into the apical dendrite. This transformation is coupled to the successful completion of migration and neuronal soma arrest occurs below the first stable branch point of the nascent dendrite. Deficiency in Reelin causes the forming dendrite to avoid its normal target area and branch aberrantly, leading to improper cellular positioning. Therefore, this study links Reelin-dependent dendritogenesis with migration arrest and cortical lamination.

A variety of fluorescent proteins have been identified that undergo shifts in spectral emission properties over time or once they are irradiated by ultraviolet or blue light. Such proteins are finding application in following the dynamics of particular proteins or labelled organelles within the cell. However, before genes encoding these fluorescent proteins were available, many proteins have already been labelled with GFP in transgenic cells; a number of model organisms feature collections of GFP-tagged lines and organisms. Here we describe a fast, localized and non-invasive method for GFP photoconversion from green to red. We demonstrate its use in transgenic plant, Drosophila and mammalian cells in vivo. While genes encoding fluorescent proteins specifically designed for photoconversion will usually be advantageous when creating new transgenic lines, our method for photoconversion of GFP allows the use of existing GFP-tagged transgenic lines for studies of dynamic processes in living cells.