Limits of Non-invasive Enzymatic Activation by Local Temperature Control.

Enzymatic activity depends on and can therefore be regulated by temperature. Selective modulation of the activity of different enzymes in one reaction pot would require temperature control local to each type of enzyme. It has been suggested previously that immobilization of enzyme on magnetic nanoparticles and exposing them to alternating magnetic field can enhance the reaction rate. This enhancement has been explained as being mediated by temperature increase caused by dissipation of the absorbed field energy in the form of heat. However, the possibility of spatially limiting this temperature increase on the microscale has been questioned. Here, it is investigated whether an activity enhancement of the enzyme sucrose phosphorylase immobilized on magnetic beads can be achieved, how this effect is related to the increase in temperature, and whether temperature differences within one reaction pot could be generated in this way. It is found that alternating magnetic field stimulation leads to increased enzymatic activity fully attributable to the increase of bulk temperature. Both theoretical analysis and experimental data indicate that no local heating near the particle surface takes place. It is further concluded that relevant increase of surface temperature can be obtained only with macroscopic, millimeter-sized, magnetic particles.

Diffusion of Single-Pass Transmembrane Receptors: From the Plasma Membrane into Giant Liposomes.

To quantitatively examine the effect of membrane organization on lateral diffusion, we studied fluorescent carbocyanine lipid analogues and EGFP-tagged, single-pass transmembrane proteins in systems of decreasing complexity: (i) the plasma membrane (PM) of living cells, (ii) paraformaldehyde/dithiothreitol-induced giant plasma membrane vesicles (GPMVs), and (iii) giant unilamellar vesicles (GUVs) under physiological buffer conditions. A truncated, signaling-deficient interleukin-4 receptor subunit, showing efficient accumulation in the plasma membrane, served as a model transmembrane protein. Two-dimensional diffusion coefficients (D) were determined by fluorescence correlation spectroscopy (FCS) either at fixed positions (single-point, spFCS) or while scanning a circular orbit (circular scanning, csFCS). Consistent with a different inclusion sizes in the membrane, lipids diffuse slightly faster than the single-spanning membrane proteins in both membrane systems, GUVs and GPMVs. In GPMVs lipids and proteins consistently experienced a fivefold larger viscosity than in GUVs, reflecting the significant fraction of plasma membrane-derived proteins partitioning into GPMVs. Lipid and protein diffusion in the PM was, respectively, 2 times and 4-5 times slower in comparison to GPMVs. This discrepancy was quantitatively confirmed by csFCS. The similarity of diffusion of receptors and lipids in GPMVs and GUVs and its significant difference in the plasma membrane suggest that protein domains as small as EGFP convey sensitivity to the actin cortex on various length scales.

MinCDE exploits the dynamic nature of FtsZ filaments for its spatial regulation.

In Escherichia coli, a contractile ring (Z-ring) is formed at midcell before cytokinesis. This ring consists primarily of FtsZ, a tubulin-like GTPase, that assembles into protofilaments similar to those in microtubules but different in their suprastructures. The Min proteins MinC, MinD, and MinE are determinants of Z-ring positioning in E. coli. MinD and MinE oscillate from pole to pole, and genetic and biochemical evidence concludes that MinC positions the Z-ring by coupling its assembly to the oscillations by direct inhibitory interaction. The mechanism of inhibition of FtsZ polymerization and, thus, positioning by MinC, however, is not understood completely. Our in vitro reconstitution experiments suggest that the Z-ring consists of dynamic protofilament bundles in which monomers constantly are exchanged throughout, stochastically creating protofilament ends along the length of the filament. From the coreconstitution of FtsZ with MinCDE, we propose that MinC acts on the filaments in two ways: by increasing the detachment rate of FtsZ-GDP within the filaments and by reducing the attachment rate of FtsZ monomers to filaments by occupying binding sites on the FtsZ filament lattice. Furthermore, our data show that the MinCDE system indeed is sufficient to cause spatial regulation of FtsZ, required for Z-ring positioning.

Introducing a fluorescence-based standard to quantify protein partitioning into membranes.

The affinity of peripheral membrane proteins for a lipid bilayer can be described using the partition coefficient (KP). Although several methods to determine KP are known, all possess limitations. To address some of these issues, we developed both: a versatile method based on single molecule detection and fluorescence imaging for determining KP, and a simple measurement standard employing hexahistidine-tagged enhanced green fluorescent protein (eGFP-His6) and free standing membranes of giant unilamellar vesicles (GUVs) functionalized with NTA(Ni) lipids as binding sites. To ensure intrinsic control, our method features two measurement modes. In the single molecule mode, fluorescence correlation spectroscopy (FCS) is applied to quantify free and membrane associated protein concentrations at equilibrium and calculate KP. In the imaging mode, confocal fluorescence images of GUVs are recorded and analyzed with semi-automated software to extract protein mean concentrations used to derive KP. Both modes were compared by determining the affinity of our standard, resulting in equivalent KP values. As observed in other systems, eGFP-His6 affinity for membranes containing increasing amounts of NTA(Ni) lipids rises in a stronger-than-linear fashion. We compared our dual approach with a FCS-based assay that uses large unilamellar vesicles (LUVs), which however fails to capture the stronger-than-linear trend for our NTA(Ni)-His6 standard. Hence, we determined the KP of the MARCKS effector domain with our FCS approach on GUVs, whose results are consistent with previously published data using LUVs. We finally provide a practical manual on how to measure KP and understand it in terms of molecules per lipid surface.

Confocal Luminescence Lifetime Imaging with Variable Scan Velocity and Its Application to Oxygen Sensing.

The dependence of the luminescence lifetime on the probe environment is the basis of a range of sensing techniques. The major advantage of using the lifetime as the sensitive parameter is its independence on the probe concentration. However, the instrumentation for lifetime measurements is complex, generally requiring time-resolved excitation and detection. Here, we present a simple method for the measurement of luminescence lifetimes on the microsecond scale based on variable excitation time determined by the scanning velocity. The technique is implemented in a confocal laser scanning microscope (CLSM), thus allowing not only simple lifetime measurement but also phosphorescence lifetime imaging. Since the method exploits the spatiotemporal dependence of sample excitation in a CLSM, there is no need for a pulsed or modulated light source or for additional time-resolved detection. The method can be realized in a standard CLSM without any modifications. The principle is demonstrated on oxygen sensing by collisional quenching of an oxygen-sensitive ruthenium(II) complex.

Let the substrate flow, not the enzyme: Practical immobilization of d-amino acid oxidase in a glass microreactor for effective biocatalytic conversions.

Exploiting enzymes for chemical synthesis in flow microreactors necessitates their reuse for multiple rounds of conversion. To achieve this goal, immobilizing the enzymes on microchannel walls is a promising approach, but practical methods for it are lacking. Using fusion to a silica-binding module to engineer enzyme adsorption to glass surfaces, we show convenient immobilization of d-amino acid oxidase on borosilicate microchannel plates. In confocal laser scanning microscopy, channel walls appeared uniformly coated with target protein. The immobilized enzyme activity was in the range expected for monolayer coverage of the plain surface with oxidase (2.37 × 10(-5) nmol/mm(2) ). Surface attachment of the enzyme was completely stable under flow. The operational half-life of the immobilized oxidase (25°C, pH 8.0; soluble catalase added) was 40 h. Enzymatic oxidation of d-Met into α-keto-γ-(methylthio)butyric acid was characterized in single-pass and recycle reactor configurations, employing in-line measurement of dissolved O2 , and off-line determination of the keto-acid product. Reaction-diffusion time-scale analysis for different flow conditions showed that the heterogeneously catalyzed reaction was always slower than diffusion of O2 to the solid surface (DaII ≤ 0.3). Potential of the microreactor for intensifying O2 -dependent biotransformations restricted by mass transfer in conventional reactors is thus revealed. Biotechnol. Bioeng. 2016;113: 2342-2349. © 2016 Wiley Periodicals, Inc.

FtsZ Polymers Tethered to the Membrane by ZipA Are Susceptible to Spatial Regulation by Min Waves.

Bacterial cell division is driven by an FtsZ ring in which the FtsZ protein localizes at mid-cell and recruits other proteins, forming a divisome. In Escherichia coli, the first molecular assembly of the divisome, the proto-ring, is formed by the association of FtsZ polymers to the cytoplasmic membrane through the membrane-tethering FtsA and ZipA proteins. The MinCDE system plays a major role in the site selection of the division ring because these proteins oscillate from pole to pole in such a way that the concentration of the FtsZ-ring inhibitor, MinC, is minimal at the cell center, thus favoring FtsZ assembly in this region. We show that MinCDE drives the formation of waves of FtsZ polymers associated to bilayers by ZipA, which propagate as antiphase patterns with respect to those of Min as revealed by confocal fluorescence microscopy. The emergence of these FtsZ waves results from the displacement of FtsZ polymers from the vicinity of the membrane by MinCD, which efficiently competes with ZipA for the C-terminal region of FtsZ, a central hub for multiple interactions that are essential for division. The coupling between FtsZ polymers and Min is enhanced at higher surface densities of ZipA or in the presence of crowding agents that favor the accumulation of FtsZ polymers near the membrane. The association of FtsZ polymers to the membrane modifies the response of FtsZ to Min, and comigrating Min-FtsZ waves are observed when FtsZ is free in solution and not attached to the membrane by ZipA. Taken together, our findings show that the dynamic Min patterns modulate the spatial distribution of FtsZ polymers in controlled minimal membranes. We propose that ZipA plays an important role in mid-cell recruitment of FtsZ orchestrated by MinCDE.

Fgf8 morphogen gradient forms by a source-sink mechanism with freely diffusing molecules.

It is widely accepted that tissue differentiation and morphogenesis in multicellular organisms are regulated by tightly controlled concentration gradients of morphogens. How exactly these gradients are formed, however, remains unclear. Here we show that Fgf8 morphogen gradients in living zebrafish embryos are established and maintained by two essential factors: fast, free diffusion of single molecules away from the source through extracellular space, and a sink function of the receiving cells, regulated by receptor-mediated endocytosis. Evidence is provided by directly examining single molecules of Fgf8 in living tissue by fluorescence correlation spectroscopy, quantifying their local mobility and concentration with high precision. By changing the degree of uptake of Fgf8 into its target cells, we are able to alter the shape of the Fgf8 gradient. Our results demonstrate that a freely diffusing morphogen can set up concentration gradients in a complex multicellular tissue by a simple source-sink mechanism.

High-resolution three-photon biomedical imaging using doped ZnS nanocrystals.

Three-photon excitation is a process that occurs when three photons are simultaneously absorbed within a luminophore for photo-excitation through virtual states. Although the imaging application of this process was proposed decades ago, three-photon biomedical imaging has not been realized yet owing to its intrinsic low quantum efficiency. We herein report on high-resolution in vitro and in vivo imaging by combining three-photon excitation of ZnS nanocrystals and visible emission from Mn(2+) dopants. The large three-photon cross-section of the nanocrystals enabled targeted cellular imaging under high spatial resolution, approaching the theoretical limit of three-photon excitation. Owing to the enhanced Stokes shift achieved through nanocrystal doping, the three-photon process was successfully applied to high-resolution in vivo tumour-targeted imaging. Furthermore, the biocompatibility of ZnS nanocrystals offers great potential for clinical applications of three-photon imaging.

Propagation of MinCDE waves on free-standing membranes.

As a spatial modulator of cytokinesis in Escherichia coli, the Min system cooperates with the nucleoid occlusion mechanism to target the divisome assembly towards mid-cell. Based on a reaction-diffusion mechanism powered by ATP (adenosine triphosphate) hydrolysis, the Min proteins propagate in waves on the cell membrane, resulting in oscillations between the cell poles, thus preventing the formation of the division ring everywhere but in the cell centre. The dynamic behaviour of Min proteins has been successfully reconstructed in vitro on supported lipid bilayers (SLBs), reproducing many of the features observed in the cell. However, there has been a marked discrepancy between the speed of propagation of Min protein waves in vitro, compared with the cellular system. A very plausible explanation is the different mobility of proteins on model membranes, compared with the inner membrane of bacteria. To quantitatively demonstrate how membrane diffusion influences Min wave propagation, we compared Min waves on SLBs with free-standing giant unilamellar vesicles (GUV) membranes which display higher fluidity. Intriguingly, the propagation velocity and wavelength on GUVs are three times higher than those reported on supported bilayers, but the wave period is conserved. This suggests that the shorter spatial period of the patterns in vivo might indeed be primarily explained by lower diffusion coefficients of proteins on the bacterial inner membrane.

Excitation spectra and brightness optimization of two-photon excited probes.

Two-photon probe excitation data are commonly presented as absorption cross section or molecular brightness (the detected fluorescence rate per molecule). We report two-photon molecular brightness spectra for a diverse set of organic and genetically encoded probes with an automated spectroscopic system based on fluorescence correlation spectroscopy. The two-photon action cross section can be extracted from molecular brightness measurements at low excitation intensities, while peak molecular brightness (the maximum molecular brightness with increasing excitation intensity) is measured at higher intensities at which probe photophysical effects become significant. The spectral shape of these two parameters was similar across all dye families tested. Peak molecular brightness spectra, which can be obtained rapidly and with reduced experimental complexity, can thus serve as a first-order approximation to cross-section spectra in determining optimal wavelengths for two-photon excitation, while providing additional information pertaining to probe photostability. The data shown should assist in probe choice and experimental design for multiphoton microscopy studies. Further, we show that, by the addition of a passive pulse splitter, nonlinear bleaching can be reduced--resulting in an enhancement of the fluorescence signal in fluorescence correlation spectroscopy by a factor of two. This increase in fluorescence signal, together with the observed resemblance of action cross section and peak brightness spectra, suggests higher-order photobleaching pathways for two-photon excitation.

Correcting for spectral cross-talk in dual-color fluorescence cross-correlation spectroscopy.

Dual-color fluorescence cross-correlation spectroscopy (dcFCCS) allows one to quantitatively assess the interactions of mobile molecules labeled with distinct fluorophores. The technique is widely applied to both reconstituted and live-cell biological systems. A major drawback of dcFCCS is the risk of an artifactual false-positive or overestimated cross-correlation amplitude arising from spectral cross-talk. Cross-talk can be reduced or prevented by fast alternating excitation, but the technology is not easily implemented in standard commercial setups. An experimental strategy is devised that does not require specialized hardware and software for recognizing and correcting for cross-talk in standard dcFCCS. The dependence of the cross-talk on particle concentrations and brightnesses is quantitatively confirmed. Moreover, it is straightforward to quantitatively correct for cross-talk using quickly accessible parameters, that is, the measured (apparent) fluorescence count rates and correlation amplitudes. Only the bleed-through ratio needs to be determined in a calibration measurement. Finally, the limitations of cross-talk correction and its influence on experimental error are explored.

Model of Processive Catalysis with Site Clustering and Blocking and Its Application to Cellulose Hydrolysis.

Interactions between particles moving on a linear track and their possible blocking by obstacles can lead to crowding, impeding the particles' transport kinetics. When the particles are enzymes processively catalyzing a reaction along a linear polymeric substrate, these crowding and blocking effects may substantially reduce the overall catalytic rate. Cellulose hydrolysis by exocellulases processively moving along cellulose chains assembled into insoluble cellulose particles is an example of such a catalytic transport process. The details of the kinetics of cellulose hydrolysis and the causes of the often observed reduction of hydrolysis rate over time are not yet fully understood. Crowding and blocking of enzyme particles are thought to be one of the important factors affecting the cellulose hydrolysis, but its exact role and mechanism are not clear. Here, we introduce a simple model based on an elementary transport process that incorporates the crowding and blocking effects in a straightforward way. This is achieved by making a distinction between binding and non-binding sites on the chain. The model reproduces a range of experimental results, mainly related to the early phase of cellulose hydrolysis. Our results indicate that the combined effects of clustering of binding sites together with the occupancy pattern of these sites by the enzyme molecules play a decisive role in the overall kinetics of cellulose hydrolysis. It is suggested that periodic desorption and rebinding of enzyme molecules could be a basis of a strategy to partially counter the clustering of and blocking by the binding sites and so enhance the rate of cellulose hydrolysis. The general nature of the model means that it could be applicable also to other transport processes that make a distinction between binding and non-binding sites, where crowding and blocking are expected to be relevant.

Circular scanning fluorescence correlation spectroscopy on membranes.

We discuss circular scanning Fluorescence Correlation Spectroscopy (sFCS) as a simple extension of standard FCS for accurate, robust and fast diffusion measurements on membranes. The implementation is based on a straightforward conversion of a conventional FCS instrument to a sFCS device by mounting a mirror onto a two-axis piezo scanner. The measurement volume is scanned in a circle with sub-micron radius, allowing the determination of diffusion coefficients and concentrations without any a priori knowledge of the size of the detection volume. This is highly important in measurements on two-dimensional surfaces, where the volume size, and therefore the quantitative outcome of the experiment, is determined by the relative position of the surface and the objective focus, a parameter difficult to control in practice. The technique is applied to diffusion measurements on model membrane systems: supported lipid bilayers and giant unilamellar vesicles. We show that the method is insensitive to membrane positioning and to disturbing processes on faster or slower time scales than diffusion, and yields accurate results even for fluctuating or drifting membranes. Its robustness, short measurement times, and small size of the probed area makes this technique particularly attractive for analyzing the properties of membranes and molecules diffusing and interacting within them.

Optimal parameters in variable-velocity scanning luminescence lifetime microscopy.

We determine the optimal parameters (scan velocities) for measuring the luminescence lifetime on the microsecond scale using the recently introduced method based on scanning the excitation beam across the sample. Using simulations, we evaluate the standard deviation and bias of the luminescence decay rate determined by scanning with two different velocities. The analysis is performed for Poisson- and normal-distributed signals, representing different types of detection techniques. We also show that a weak uncorrected background induces a bias in the obtained decay rate, and take this effect into account when choosing optimal measurement parameters. For comparison, the analysis is additionally performed for two conventional gating schemes for lifetime measurement. The variable-velocity scanning method is found to be more robust to the effect of the background signal than the gating schemes.

Photon arrival timing with sub-camera exposure time resolution in wide-field time-resolved photon counting imaging.

We demonstrate that an ultra-fast CMOS camera combined with a photon counting image intensifier can be used to determine photon arrival times well below the exposure time of the camera. We can obtain a time resolution down to around 1% of the exposure time, i.e. of the order of 40 ns with microsecond exposure times. This is achieved by exploiting the invariant phosphor decay of the image intensifier's phosphor screen: Developing a suitable mathematical framework, we show that the relative intensities of the phosphor decay in successive frames following the photon detection uniquely determine the photon arrival time. This approach opens a way to measuring fast luminescence decays in parallel in many pixels. Possible applications include oxygen and ion concentration imaging using probes with luminescence lifetimes in the range of 100 ns to microseconds.

Intraparticle pH Sensing Within Immobilized Enzymes: Immobilized Yellow Fluorescent Protein as Optical Sensor for Spatiotemporal Mapping of pH Inside Porous Particles.

pH is a fundamental variable in enzyme catalysis and its measurement therefore is crucial for understanding and optimizing enzyme-catalyzed reactions. Whereas measurements within homogeneous bulk liquid solution are prominently used, enzymes immobilized inside porous particles often suffer from pH gradients due to partition effects and heterogeneously catalyzed biochemical reactions. Unfortunately, the measurements of intraparticle pH are not available due to the lack of useful suitable methodologies; as a consequence the biocatalyst characterization is hampered. Here, a fully biocompatible methodology for real-time optical sensing of pH within porous materials is described. A genetically encoded ratiometric pH indicator, the superfolder yellow fluorescent protein (sYFP), is used to functionalize the internal surface of enzyme carrier supports. By using controlled, tailor-made immobilization, sYFP is homogeneously distributed within these materials, and so enables, via self-referenced imaging analysis, pH measurements in high accuracy and with useful spatiotemporal resolution. The hydrolysis of penicillin by a penicillin acylase, taking place in solution or confined to the solid surface of the porous matrix is used to show the monitoring of evolution of internal pH. Thus, pH sensing based on immobilized sYFP represents a broadly applicable technique to the study of the internally heterogeneous environment of immobilized enzymes into solid particles.

Wide-field photon counting fluorescence lifetime imaging microscopy: application to photosynthesizing systems.

Fluorescence lifetime imaging microscopy (FLIM) is a technique that visualizes the excited state kinetics of fluorescence molecules with the spatial resolution of a fluorescence microscope. We present a scanningless implementation of FLIM based on a time- and spacecorrelated single photon counting (TSCSPC) method employing a position-sensitive quadrant anode detector and wide-field illumination. The standard time-correlated photon counting approach leads to picosecond temporal resolution, making it possible to resolve complex fluorescence decays. This allows parallel acquisition of time-resolved images of biological samples under minimally invasive low-excitation conditions (<10 mW/cm(2)). In this way unwanted photochemical reactions induced by high excitation intensities and distorting the decay kinetics are avoided. Comparably low excitation intensities are practically impossible to achieve with a conventional laser scanning microscope, where focusing of the excitation beam into a tight spot is required. Therefore, wide-field FLIM permits to study Photosystem II (PS II) in a way so far not possible with a laser scanning microscope. The potential of the wide-field TSCSPC method is demonstrated by presenting FLIM measurements of the fluorescence dynamics of photosynthetic systems in living cells of the chlorophyll d-containing cyanobacterium Acaryochloris marina.

Wide-field time-correlated single photon counting-based fluorescence lifetime imaging microscopy.

Wide-field time-correlated single photon counting detection techniques, where the position and the arrival time of the photons are recorded simultaneously using a camera, have made some advances recently. The technology and instrumentation used for this approach is employed in areas such as nuclear science, mass spectroscopy and positron emission tomography, but here, we discuss some of the wide-field TCSPC methods, for applications in fluorescence microscopy. We describe work by us and others as presented in the Ulitima fast imaging and tracking conference at the Argonne National Laboratory in September 2018, from phosphorescence lifetime imaging (PLIM) microscopy on the microsecond time scale to fluorescence lifetime imaging (FLIM) on the nanosecond time scale, and highlight some applications of these techniques.

Precise measurement of diffusion coefficients using scanning fluorescence correlation spectroscopy.

We have implemented scanning fluorescence correlation spectroscopy (sFCS) for precise determination of diffusion coefficients of fluorescent molecules in solution. The measurement volume where the molecules are excited, and from which the fluorescence is detected, was scanned in a circle with radius comparable to its size at frequencies 0.5-2 kHz. The scan radius R, determined with high accuracy by careful calibration, provides the spatial measure required for the determination of the diffusion coefficient D, without the need to know the exact size of the measurement volume. The difficulties in the determination of the measurement volume size have limited the application of standard FCS with fixed measurement volume to relative measurements, where the diffusion coefficient is determined by comparison with a standard. We demonstrate, on examples of several common fluorescent dyes, that sFCS can be used to measure D with high precision without a need for a standard. The correct value of D can be determined in the presence of weak photobleaching, and when the measurement volume size is modified, indicating the robustness of the method. The applicability of the presented implementation of sFCS to biological systems in demonstrated on the measurement of the diffusion coefficient of eGFP in the cytoplasm of HeLa cells. With the help of simulations, we find the optimal value of the scan radius R for the experiment.