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In this work, a deep learning-based method, STED-flimGANE, is introduced to achieve enhanced STED imaging resolution under a low STED-beam power and photon-starved conditions.

A series of tuneable emission phosphors Ba2Ca1-xMx(PO4)2:Eu2+ (M = Mg, Sr, Mg–Sr, and Zn) was prepared using multi-cation substitution. A new mechanism of direct/indirect substitution of Eu2+ for a host containing more than one cation and a concept of “selective excitation” for a multiple luminescence centres are proposed that are of great significance for analysing the changes of spectra and for designing colour tuneable phosphors. The emission spectra show a red shift (479 → 602 nm) with colour tuning from blue to yellow in Ba2Ca1-xMgx(PO4)2:Eu2+ and a blue shift (485 → 441 nm) in Ba2Ca1-xSrx(PO4)2:Eu2+. For Ba2Ca1-xZnx(PO4)2:Eu2+, the emission peak first turns from 494 to 442 nm and then a red shift occurs from 442 to 585 nm when excited at 355 nm, while a continuous red shift was observed under excitation of 400 nm. Three different patterns (solid solution material, mixture transition state, and new host component) of phosphors were presented when many foreign cations were introduced into the original host. The energy transfer and thermal back-transfer over the barrier were demonstrated in as-prepared samples. Warm white light-emitting diodes (LEDs) with the International Commission on Illumination (CIE) chromaticity coordinates of (0.3622 and 0.3051), a colour rendering index (Ra) of 88.7, and a correlated colour temperature (CCT) of 3947 were realised using as-prepared phosphors. The as-developed compound series has the potential for further optimisation for commercial applications in daylight LEDs and warm white LEDs.

The ongoing COVID‐19 pandemic caused by the SARS‐CoV‐2 coronavirus has affected the health of tens of millions of people worldwide. In particular, in elderly and frail individuals the infection can lead to severe disease and even fatal outcomes. Although the pandemic is primarily a human health crisis its consequences are much broader with a tremendous impact on global economics and social systems. Vaccines are considered the most powerful measure to fight the pandemic and protect people from COVID‐19. Based on the concerted activities of scientists, manufacturers and regulators, the urgent need for effective countermeasures has provoked the development and licensure of novel COVID‐19 vaccines in an unprecedentedly fast and flexible manner within

Forster resonance energy transfer (FRET) microscopy is a technique that provides the spatial and temporal resolution necessary to investigate protein-protein interactions in live specimens. One of the most robust ways of quantifying FRET is to measure the fluorescence lifetime of the donor fluorophore. Fluorescence lifetime imaging microscopy (FLIM) is routinely used in the biological sciences to monitor dynamic signaling events inside living cells. However, the demands of studying complex signaling networks in living cells using FRET-based biosensor probes requires the ability to track more than one donor fluorophore at the same time. Here, we demonstrate a novel (Pulsed Interleave Excitation) PIE-FastFLIM technique that can be used to simultaneously measure two FRET-based biosensor probes at the same pixel. The PIE-FastFLIM configuration uses tunable wavelengths and synchronized gating detection, which can be tailored and optimized for each biosensor probe. When combined with the model-free phasor plots analysis, the PIEFastFLIM approach enables quantitative multiplexed FRET measurements for tracking multiple signaling events simultaneously in live cells.

Multiple preventive COVID-19 vaccines have been developed during the ongoing SARS coronavirus (CoV) 2 pandemic, utilizing a variety of technology platforms, which have different properties, advantages, and disadvantages. The acceleration in vaccine development required to combat the current pandemic is not at the expense of the necessary regulatory requirements, including robust and comprehensive data collection along with clinical product safety and efficacy evaluation. Due to the previous development of vaccine candidates against the related highly pathogenic coronaviruses SARS-CoV and MERS-CoV, the antigen that elicits immune protection is known: the surface spike protein of SARS-CoV-2 or specific domains encoded in that protein, e.g., the receptor binding domain. From a scientific point of view and in accordance with legal frameworks and regulatory practices, for the approval of a clinic trial, the Paul-Ehrlich-Institut requires preclinical testing of vaccine candidates, including general pharmacology and toxicology as well as immunogenicity. For COVID-19 vaccine candidates, based on existing platform technologies with a sufficiently broad data base, pharmacological–toxicological testing in the case of repeated administration, quantifying systemic distribution, and proof of vaccination protection in animal models can be carried out in parallel to phase 1 or 1/2 clinical trials. To reduce the theoretical risk of an increased respiratory illness through infection-enhancing antibodies or as a result of Th2 polarization and altered cytokine profiles of the immune response following vaccination, which are of specific concern for COVID-19 vaccines, appropriate investigative testing is imperative. In general, phase 1 (vaccine safety) and 2 (dose finding, vaccination schedule) clinical trials can be combined, and combined phase 2/3 trials are recommended to determine safety and efficacy. By applying these fundamental requirements not only for the approval and analysis of clinical trials but also for the regulatory evaluation during the assessment of marketing authorization applications, several efficacious and safe COVID-19 vaccines have been licensed in the EU by unprecedentedly fast and flexible procedures. Procedural and regulatory–scientific aspects of the COVID-19 licensing processes are described in this review.

The mesoscopic scale is between microscopic and macroscopic scales. In life sciences, mesoscopic imaging allows scientists to record, track and study details of biological systems in the context of an organ, body part, or organism. Mesoscopic imaging techniques have been developed for medical and clinical research, such as drug delivery, cancer diagnosis, etc. Especially when combined with novel nanoparticles and organic dyes in the near-infrared spectral regime, the mesoscopic imaging can probe deeper parts of the animal body. Here, we describe a timeresolved mesoscopic imaging approach, which can image deep inside of the whole mouse noninvasively. In addition, it uses the FastFLIM technique to measure the lifetime of the fluorescent probe. Since the lifetime carries information about the probe’s local microenvironment such as temperature, pH, ion concentration, etc., the lifetime imaging map obtained by the FastFLIM-mesoscope allows tracking quantitative dynamics of the probes in the whole animal body. The technique can also be used for quantitative intrinsic NADH metabolism mapping for real time monitoring of mitochondrial function. Here, we will show mesoscopic-scale NADH imaging in an oral cancer model.

We present a microscopy technique, orbital particle tracking, in which the scanner scans orbits around species, unlike a raster imaging technique in which the scanner scans an area one line at a time. By analyzing the fluorescence emission intensity variation along an orbit, the location of a species in the orbit can be determined with precision of a tenth of a nanometer in a millisecond time scale, and the orbit can be moved to the new location of the species through a feedback loop if any movement is detected. This technique can be extended to two scanning orbits, one above and one below the sample plane to track the sample in 3D space. It can be used in vitro or in vivo to track a motion of a sample or to understand the dynamics of the sample. Additional detectors can help reveal the correlation between events with different emission spectrums. We have performed two different experiments with the system to show the capability of the technique. In the first example, we track a transcription site to understand the relationship between transcription factor - DNA binding and RNA transcription [1, 2]. By labeling a transcription factor with Halo-JF646 and nascent RNA with PP7-GFP, we were able to cross correlate fluorescence intensity to discover temporal coordination between transcription factor DNA binding and resulting gene activation. In the second experiment, we tracked lysosomes in live cells to understand the nature of the transport whether it is an active transport or a free diffusion [3]. Trajectories of a total of 24 lysosomes are recorded during the experiment. The mean squared displacement (MSD) curves of the trajectories showed some clear differences between the behaviors of the lysosomes which were attributed to the active transport along microtubules as opposed to freely diffusing lysosomes.

Summary Emerging all-inorganic perovskite nanocrystals can retain a desired crystal structure under ambient conditions and offer easy solution processability. In this work, we have demonstrated CsPbI3 perovskite quantum dot (QD) solar cells with a remarkable efficiency approaching 13% and an extremely low energy loss of 0.45 eV by employing a series of dopant-free polymeric hole-transporting materials (HTMs). The CsPbI3 QD solar cells use polymer HTMs to achieve efficient charge extraction at QD/polymer interfaces and avoid device instability caused by complex doping and oxidation processes required by conventional Spiro-OMeTAD. Meanwhile, the CsPbI3 QD photovoltaic devices can be fabricated at room temperature and exhibit more reproducible film quality, showing potential advantages over current all-inorganic thin-film perovskite solar cells. We believe that our findings will catalyze the development of new device structures, specifically for perovskite QDs, and help realize the promising potential of all-inorganic perovskite solar cells.

Background To assess the safety and efficacy of a novel technology referred to as percutaneous ureteroscopic plasma column electrode (PCE) by comparing laparoscopic decortication in the management of simple renal cyst (SRC). Methods Between March 2016 and June 2017, 53 patients with SRCs were randomized to divided into two groups, the PCE group (24 patients), or laparoscope group (29 patients). The operative time, blood loss, days of drainage, catheter, and hospital stay and complications were compared with the two groups. All patients were followed- up to 6 months after treatment. Results No patients had intraoperative complications such as hemopneumothorax, adjacent organ injury, infection or hemorrhage shock. In the PCE group and laparoscope group: the mean operation time was 34.1±8.2 vs. 58.4±16.7 min (P0.05). The mean intra-urethral indwelling catheter time was 2.1±0.88 vs. 2.0±1.15 d (P>0.05). The mean postoperative hospital stay was 3.0±1.7 vs. 3.7±1.53 (P>0.05). One patient in electrode group was suffered from rupture of the collecting system during the operation, and was treated by indwelling D-J stent. During follow up, no cysts recurrence was found. Conclusions Percutaneous ureteroscopic PCE decortication is a safe, minimally invasive and effective therapy to treat SRCs, with equal efficacy and advantages in shortening the operation time and reducing the amount of intraoperative bleeding compared with laparoscopic decortication.

We report three synthetic methods to prepare biocompatible Yb3+ complexes, which displayed high NIR luminescence with quantum yields up to 13% in aqueous media. This renders β-fluorinated Yb3+ porphyrinoids a new class of NIR probes for living cell imaging including time-resolved fluorescence lifetime imaging., Herein, we report the design and synthesis of biocompatible Yb3+ complexes for near-infrared (NIR) living cell imaging. Upon excitation at either the visible (Soret band) or red region (Q band), these β-fluorinated Yb3+ complexes display high NIR luminescence (quantum yields up to 23% and 13% in dimethyl sulfoxide and water, respectively) and have higher stabilities and prolonged decay lifetimes (up to 249 μs) compared to the β-non-fluorinated counterparts. This renders the β-fluorinated Yb3+ complexes as a new class of biological optical probes in both steady-state imaging and time-resolved fluorescence lifetime imaging (FLIM). NIR confocal fluorescence images showed strong and specific intracellular Yb3+ luminescence signals when the biocompatible Yb3+ complexes were uptaken into the living cells. Importantly, FLIM measurements showed an intracellular lifetime distribution between 100 and 200 μs, allowing an effective discrimination from cell autofluorescence, and afforded high signal-to-noise ratios as firstly demonstrated in the NIR region. These results demonstrated the prospects of NIR lanthanide complexes as biological probes for NIR steady-state fluorescence and time-resolved fluorescence lifetime imaging.

High luminous efficacy, high-color-rendering index (CRI), single-phased and single Eu2+ doped warm white-light phosphors Ca2(BO3)1−x(PO4)xCl were synthesized by controlling anion substitution systematically. The anionic polyhedron substitution of PO4 for BO3 causes a great distortion of the host lattice, which enables the tuning of luminescent properties. An additional distinct emission peaking at 462 nm (in addition to 577 nm of Ca2BO3Cl:Eu2+) was observed under the same excitation when PO43− was introduced. Therefore, with the crystal structure evolution, the ratio blue/yellow was changed by adjusting the ratio of BO3/PO4, which provides a new way for developing a high CRI warm white-light phosphor. The internal quantum efficiency (IQE) of the Ca2(BO3)0.64(PO4)0.36Cl:Eu2+ phosphor was 64.0%. The warm white light-emitting diode (w-LED) was fabricated with Ca2(BO3)0.64(PO4)0.36Cl:Eu2+ phosphor and a 380 nm-emitting InGaN chip, which exhibited promising properties, such as the high Ra (83.4), the low correlated color temperature (3907 K) and high luminous efficacy ɳL (30.4 lm/W). Importantly, the w-LED shows a greater R9 = 39.3 versus R9 = 14.3 of commercial YAG:Ce3+. These phenomenon demonstrates that Ca2(BO3)1−x(PO4)xCl:Eu2+ as a warm white-light phosphor will have a great prospect of application in indoor lighting.

Generally, the luminescence properties of phosphors can be tuned by cations substitutions (Mg2+, Ca2+, Sr2+ and Ba2+); however, the underlying mechanism is not clear. The luminescence properties are influenced by many factors, such as symmetry of the ligand polyhedron and the bond length between the luminescent centre and its ligand. Therefore, in this research, the phosphor Ca9−x−y−zMgxSryBazCe(PO4)7:Eu2+,Mn2+ was selected as the research object, and the reasons are investigated. Detailed data regarding the crystal space structure is acquired through the Rietveld refinement and used to systematically analyze the change of the two factors. It was found that the two factors are sometimes synergetic and sometimes antagonistic to the spectrum shift. The change of the luminescence properties is the comprehensive result of the two factors. It was found that the influence produced by the change of symmetry of the polyhedron is stronger than that by the change of bond length between the luminescence centre and its ligand. When changing the doping concentration of Sr2+ and Ba2+, the chromaticity coordinates of (Ca,Sr,Ba)8.78Ce(PO4)7:Eu2+,Mn2+ could change from cold white light to warm white light, and there is a larger distribution range in the white light region in the CIE 1931 chromaticity diagram. Cations substitutions tuning the luminescence thermal stability are also observed. When partial Ca2+ are substituted by other cations, the luminescence thermal stability of the luminescent centre was improved to some degree. It is expected that these methods can be generalized to analyze the changes in optical and other properties that are sensitive to local coordination environments for luminescent materials.

A series of near-infrared (NIR) emitting Li2ZnGe3O8 (LZG):xMn2+ phosphors were synthesized via a conventional solid state reaction. They can be obtained by controlling the heating temperature (950 °C) or changing the doping concentration of Mn2+ (900 °C). In particular, due to the transition of the crystal from ZnO6 to ZnO4, the NIR phosphors perform with different spectral widths when the Zn/Mg ratio in LZG:xMn2+ is adjusted. The excitation spectrum is characterized by a broad band from 240 nm to 640 nm, centered at 475 nm and tailing on the low energy side. The emission spectrum presents a broad emission band from 650 to 900 nm with the main peak at 832 nm. Hence, the phosphors can convert visible and ultraviolet light to NIR emission. What's more, this kind of NIR phosphor is based on the mechanism of down-conversion, and it possesses excellent thermal effect properties.

Single molecule imaging techniques allow tracking dynamic behaviors of individual molecules, providing insight information of molecular processes that could be hidden by the ensemble average. Combining with the time-resolved imaging capability, the laser scanning confocal microscopy at the single molecule sensitivity allows quantitative multiparameter analyses of single molecule dynamics. Here, we describe the single molecule imaging tools in the ISS VistaVision software, including FCS, FCCS, PIE-FCCS, FLCS, PCH, FLIM, steady-state and time-resolved FRET, steady-state and time-resolved polarization anisotropy, burst analysis for single molecule FRET and stoichiometry, antibunching. We demonstrate with measurement examples how these techniques are used for studying photophysical properties and behaviors of single molecules, such as diffusion rates, molecular brightness, triplet time, rotational relaxation time, fluorescence lifetime. By using donor-acceptor FRET pair-labeled proteins, we detect changes in protein conformation and dynamics by quantitatively measuring FRET efficiency, stoichiometry and lifetime. This quantitative multi-parameter analysis approach gives researchers more opportunities for a better understanding of single molecule dynamics.

Stimulated emission depletion microscopy (STED) is one of the pivotal super-resolution techniques. It overcomes the spatial resolution limit imposed by the diffraction by using an additional laser beam, the STED beam, intensity of which is directly related to the achievable resolution. Despite reaching nanometer resolution, much effort in recent years has been devoted to reducing the STED beam intensity because it may lead to photo-damaging effects. Accessing the spatial information encoded in the temporal dynamics of the detected fluorescent photons has been proved to be a powerful strategy and has contributed to the separation by lifetime tuning (SPLIT) technique. The SPLIT method uses the phasor analysis to efficiently distinguish photons emitted from the center and the periphery of the excitation spot. It thus improves the resolution without increasing the STED beam intensity. This method was proposed for architectures based on the STED beam running in continuous waves (CW-STED microscopy). Here, we extend it to pulsed STED beam implementations (pSTED microscopy). We show, through simulated and experimental data, that the pSTED-SPLIT method reduces the detection volume of the pSTED microscope without significantly decreasing the signal-to-noise ratio of the final image, thus effectively improving the resolution without increasing the STED beam intensity.

Stimulated emission depletion (STED) microscopy is a powerful super-resolution microscopy technique that enables observation of macromolecular complexes and sub-cellular structures with spatial resolution well below the diffraction limit. However, resolution in the double-digit nanometer range can be obtained only using high intensity depletion laser, at the cost of increased photo-damage, which significantly limits STED applications in live specimens. To minimize this, we use the separation by lifetime tuning (SPLIT) technique, in which phasor analysis is used to efficiently distinguish photons emitted from the center and from the periphery of the excitation spot of a STED microscope. Thus, it can be used to improve the resolution without increasing the STED beam intensity. Our approach utilizes a combination of pulsed excitation and pulsed depletion lasers to record the time-resolved photons by FastFLIM. The photons stream are successively analyzed using the SPLIT technique, demonstrating that the resolution improves without increasing the depletion laser intensity.

A series of Ca0.75Sr0.2Mg1.05(Si2O6) (CSMS):xEu2+, yMn2+ phosphors were synthesized by a high temperature solid state reaction technique in a reducing atmosphere (1250 °C, 4 h). Under excitation of 270 nm, the emission spectrum of CSMS host is characterized by a broad band with two distinct emission peaks (453 and 580 nm), tailing on the energy side from approximately 350 to 800 nm. Thus, there is an orange-yellow emission in CSMS host. The different trap depths of thermoluminescence spectra (E1 = 0.6453 eV, E2 = 0.8271 eV) are measured and these traps will fade away when Eu2+ or Mn2+ ions are doped. In addition, the blue-shift, red-shift, and intensity changes of temperature dependence emission spectra in Ca0.75Sr0.2Mg1.05(Si2O6) host are observed with higher temperature. For Eu2+ single doped CSMS, which has an obvious absorption in the near-ultraviolet and blue region with a wide band of excitation spectrum ranging from 200 to 450 nm monitored at 453 nm. And the emission spectrum presents an obvious re...

A series of new tunable emission phosphors of Sm3+ and Eu3+ single-doped M3Tb(BO3)3 (M = Sr and Ba) phosphors were synthesized by the solid-state reaction. Crystallization behavior and structure, reflectance spectral properties, luminescence properties, energy transfer, lifetimes, temperature-dependent luminescence properties and CIE chromaticity coordinate of M3Tb(BO3)3:Ln3+ (M = Sr and Ba, Ln = Sm and Eu) were systematically investigated. M3Tb(BO3)3 (M = Sr and Ba) crystallizes in a rhombohedral cell with space group R by Rietveld structure refinement of the obtained phosphors with the standard data of Ba3Dy(BO3)3. Sr3Tb(BO3)3 and Ba3Tb(BO3)3 emit yellowish-green emission with the main peak around 555 and 550 nm, respectively, which originates from the 5D4 → 7F4 transition of Tb3+, and M3Tb(BO3)3:Ln3+ (Ln = Sm and Eu) phosphors show intense yellowish-green, yellow, orange and red emission with increasing Ln3+ concentration under 274 and 286 nm excitation. The orange-red/red emissions show peak maxima at 613 nm (Sr3Tb(BO3)3:Sm3+), 627 nm (Sr3Tb(BO3)3:Eu3+), 607 nm (Ba3Tb(BO3)3:Sm3+) and 625 nm (Ba3Tb(BO3)3:Eu3+), and are due to the efficient energy transfer of Tb3+–Sm3+ and Tb3+–Eu3+. The temperature-dependent luminescence properties of M3Tb(BO3)3:Ln3+ are characterised by good thermal stabilities up to 150 °C, up to 72.8%. However, due to the defect in the host of the Sr3Tb0.99(BO3)3:0.01Sm3+ phosphor, an amazing and interesting phenomenon can be observed that the emission intensity is enhanced constantly with increasing temperature.

A series of Ca9−x−yCe(PO4)7:xEu2+,yMn2+ phosphors were synthesized by a high-temperature solid-state reaction method. The as-prepared samples were characterized by XRD and EDX measurements, which showed that Eu2+ and Mn2+ could be efficiently doped into the host. Ce3+ acts concurrently as activator and sensitizer in Ca9Ce(PO4)7, and the energy transfer mechanisms between Ce3+/Eu2+ and Ce3+/Mn2+ in Ca9Ce(PO4)7 were validated and proven to be a resonant type via dipole–quadrupole and dipole–dipole interactions, respectively. Besides, there is also energy transfer from Eu2+ to Mn2+ ions. The host, Ca9Ce(PO4)7, emits blue-white light and Ca9Ce(PO4)7:xEu2+,yMn2+ phosphors emit blue-green through white to orange-red light under near-ultraviolet radiation as a result of tuning the ratio of Eu2+/Mn2+. Ca9Ce(PO4)7:0.04Eu2+,0.08Mn2+ emits white light with CIE coordinates (0.333, 0.310), a CCT of 5446 K, and a high CRI of 81. The energy transfer efficiency between Ce3+ and Mn2+ increases significantly with temperature. These results reveal that Ca9Ce(PO4)7:Eu2+,Mn2+ may be a potential candidate for white light-emitting phosphors.

In order to meet the real-time requirement of bearing fault diagnosis, a simplified strategy of the traditional long short term memory (LSTM) neural network structure is proposed. We designed a new single gated unite (SGU) recurrent neural network. In view of the non-stationary and non-linear characteristics of bearing fault vibration data, we use wavelet packet decomposition to extract the features as input signals of bi-directional single gated unite (Bi-SGU) to complete the diagnosis of 10 types of bearing data. Simulation results show that the proposed method can ensure the accuracy of bearing fault diagnosis, reduce the number of network parameters by 36%, and improve time efficiency by 41%.

Photon upconversion is a nonlinear process in which the sequential of absorption of two or more photons leads to the anti-stoke emission. Different than the conventional multiphoton excitation process, upconversion can be efficiently performed at low excitation densities. Recent developments in lanthanide-doped upconversion nanoparticles (UCNPs) have led to a diversity of applications, including detecting and sensing of biomolecules, imaging of live cells, tissues and animals, cancer diagnostic and therapy, etc. Measuring the upconversion lifetime provides a new dimension of its imaging and opens a new window for its applications. Due to the long metastable intermediate excited state, UCNP typically has a long excited state lifetime ranging from sub-microseconds to milliseconds. Here, we present a novel development using the FastFLIM technique to measure UCNP lifetime by laser scanning confocal microscopy. FastFLIM is capable of measuring lifetime from 100 ps to 100 ms and features the high data collection efficiency (up to 140-million counts per second). Other than the traditional nonlinear least-square fitting analysis, the raw data acquired by FastFLIM can be directly processed by the model-free phasor plots approach for instant and unbiased lifetime results, providing the ideal routine for the UCNP photoluminescence lifetime microscopy imaging.

Photoluminescence (PL) refers to light emission initiated by any form of photon excitation. PL spectroscopy and microscopy imaging has been widely applied in material, chemical and life sciences. Measuring its lifetime yields a new dimension of the PL imaging and opens new opportunities for many PL applications. In solar cell research, quantification of the PL lifetime has become an important evaluation for the characteristics of the Perovskite thin film. Depending upon the PL process (fluorescence, phosphorescence, photon upconversion, etc.), the PL lifetimes to be measured can vary in a wide timescale range (e.g. from sub-nanoseconds to microseconds or even milliseconds) – it is challenging to cover this wide range of lifetime measurements by a single technique efficiently. Here, we present a novel digital frequency domain (DFD) technique named FastFLIM, capable of measuring the PL lifetime from 100 ps to 100 ms at the high data collection efficiency (up to 140-million counts per second). Other than the traditional nonlinear leastsquare fitting analysis, the raw data acquired by FastFLIM can be directly processed by the model-free phasor plots approach for instant and unbiased lifetime results, providing the ideal routine for the PL lifetime microscopy imaging.

Experiments using live cell 3-color Forster (or fluorescence) resonance energy transfer (FRET) microscopy and corresponding in vitro biochemical reconstitution of the same proteins were conducted to evaluate actin filament nucleation. A novel application of 3-color FRET data is demonstrated, extending the analysis beyond the customary energy-transfer efficiency (E%) calculations. MDCK cells were transfected for coexpression of Teal-N-WASP/Venus-IQGAP1/mRFP1-Rac1, Teal-N-WASP/Venus-IQGAP1/mRFP1-Cdc42, CFP-Rac1/Venus-IQGAP1/mCherry-actin, or CFP-Cdc42/Venus-IQGAP1/mCherry-actin, and with single-label equivalents for spectral bleedthrough correction. Using confirmed E% as an entry point, fluorescence levels and related ratios were correlated at discrete accumulating levels at cell peripheries. Rising ratios of CFP-Rac1:Venus-IQGAP1 were correlated with lower overall actin fluorescence, whereas the CFP-Cdc42:Venus-IQGAP1 ratio correlated with increased actin fluorescence at low ratios, but was neutral at higher ratios. The new FRET analyses also indicated that rising levels of mRFP1-Cdc42 or mRFP1-Rac1, respectively, promoted or suppressed the association of Teal-N-WASP with Venus-IQGAP1. These 3-color FRET assays further support our in vitro results about the role of IQGAP1, Rac1, and Cdc42 in actin nucleation, and the differential impact of Rac1 and Cdc42 on the association of N-WASP with IQGAP1. In addition, this study emphasizes the power of 3-color FRET as a systems biology strategy for simultaneous evaluation of multiple interacting proteins in individual live cells. © 2015 International Society for Advancement of Cytometry

Phosphorescence probes can have significantly long lifetimes, on the order of micro- to milli-seconds or longer. In addition, environmental changes can affect the lifetimes of these phosphorescence probes. Thus, Phosphorescence Lifetime Imaging Microscopy (PLIM) is a very useful tool to localize the phosphorescence probes based on their lifetimes to study the variance in the lifetimes due to the micro environmental changes. Since the probes respond to the biologically relevant parameters like oxygen concentration, they can be used to study various biologically relevant processes like cellular metabolism, protein interaction etc. In this case, we study the effects of oxygen on Oxyphor G4 with PLIM. Since The Oxyphor G4 can be quenched by O2, it is a good example of such a probe and has a lifetime around 250us. Here we present the digital frequency domain PLIM technique and study the lifetime of the Oxyphor G4 as a function of the O2 concentration. The lifetime data are successfully presented in a phasor plot for various O2 concentrations and are consistent with the time domain data. Overall, we can analyze the oxygen consumption of varying cells using this technique.

PIE FastFLIM microscopy allows the quantitative multi-parameter measurement of single molecule protein folding and dynamics. Using donor-acceptor FRET pair-labeled proteins, we detect changes in protein conformation and dynamics by monitoring FRET efficiency, stoichiometry and lifetime. Together with anisotropy decay information, we acquire rotational relaxation times for single molecules. By applying antibunching, FLCS and burst analysis, multi-parameters (such as copy numbers in protein complexes), diffusion coefficient and molecular brightness can be fitted for deeper understanding of the conformational dynamic behavior of single protein molecules. In this paper, we’ll focus on the multiparameters of FRET efficiency, stoichiometry and lifetime.

Stimulated emission depletion (STED) microscopy is a powerful super-resolution microscopy technique that enables observation of macromolecular complexes and sub-cellular structures with spatial resolution below the diffraction limit. The spatial resolution of STED is limited by power of the depletion laser at the specimen plane. Higher depletion laser power will improve resolution, but at the cost of increased photo-bleaching, photo-toxicity, and anti-stoke emission background. This degrades the signal-to-noise ratio, and can significantly limit STED applications in living specimens. Here, we present an efficient multi-color STED microscopy method based on the digital frequency domain fluorescence lifetime imaging (FastFLIM) and the phasor plots. Our approach utilizes a combination of pulsed excitation and pulsed depletion lasers to record the time-resolved photons by FastFLIM. We demonstrate that the resolution is improved without increasing the depletion laser power by digital separation of the depleted species from the partially depleted species based on their different decay kinetics. We show the utility of this novel STED method applied in both fixed and live cellular samples, and also show its application to fluorescence lifetime correlation spectroscopy (FLCS) measurements. By combining fluorophores with different fluorescence lifetimes, we simultaneously record two-color STED images of cells labeled with Atto655 and Alexa647 in a single scan by using a single pair of excitation and depletion lasers. This novel approach shortens the data acquisition time while minimizing the photo-toxicity caused when using two separate depletion lasers.

IQGAP1 stimulates branched actin filament nucleation by activating N-WASP, which then activates the Arp2/3 complex. N-WASP can be activated by other factors, including GTP-bound Cdc42 or Rac1, which also bind IQGAP1. Here we report the use of purified proteins for in vitro binding and actin polymerization assays, and Forster (or fluorescence) resonance energy transfer (FRET) microscopy of cultured cells to illuminate functional interactions among IQGAP1, N-WASP, actin, and either Cdc42 or Rac1. In pyrene-actin assembly assays containing N-WASP and Arp2/3 complex, IQGAP1 plus either small G protein cooperatively stimulated actin filament nucleation by reducing the lag time before 50% maximum actin polymerization was reached. Similarly, Cdc42 and Rac1 modulated the binding of IQGAP1 to N-WASP in a dose-dependent manner, with Cdc42 enhancing the interaction and Rac1 reducing the interaction. These in vitro reconstitution results suggested that IQGAP1 interacts by similar, yet distinct mechanisms with Cdc42 versus Rac1 to regulate actin filament assembly through N-WASP in vivo. The physiological relevance of these multi-protein interactions was substantiated by 3-color FRET microscopy of live MDCK cells expressing various combinations of fluorescent N-WASP, IQGAP1, Cdc42, Rac1, and actin. This study also establishes 3-color FRET microscopy as a powerful tool for studying dynamic intermolecular interactions in live cells.

The fundamental theory of Forster resonance energy transfer (FRET) was established in the 1940s. Its great power was only realized in the past 20 years after different techniques were developed and applied to biological experiments. This success was made possible by the availability of suitable fluorescent probes, advanced optics, detectors, microscopy instrumentation, and analytical tools. Combined with state-of-the-art microscopy and spectroscopy, FRET imaging allows scientists to study a variety of phenomena that produce changes in molecular proximity, thereby leading to many significant findings in the life sciences. In this review, we outline various FRET imaging techniques and their strengths and limitations; we also provide a biological model to demonstrate how to investigate protein-protein interactions in living cells using both intensity- and fluorescence lifetime-based FRET microscopy methods.

A series of Ca

The crosstalk between two fluorescent species causes problems in fluorescence microscopy imaging, especially for quantitative measurements such as co-localization, Forster resonance energy transfer (FRET), fluorescence cross correlation spectroscopy (FCCS). In laser scanning confocal microscopy, the lasers can be switched on and off by acousto-optic tunable filters (AOTF) in the microsecond scale for alternative line scanning in order to avoid the crosstalk while minimizing the time delay between two lasers on the same pixel location. In contrast, the pulsed interleaved excitation (PIE) technique synchronizes two pulsed lasers of different wavelengths in the nanosecond scale to enable measuring superfast dynamics of two fluorescent species simultaneously and yet quantitatively without the crosstalk contamination. This feature is critical for many cell biology applications, e.g. accurate determination of stoichiometry in FRET measurements for studying protein-protein interactions or cell signal events, detection of weaker bindings in FCCS by eliminating the false cross correlation due to the crosstalk. The PIE has been used with the time correlated single photon counting (TCSPC) electronics. Here, we describe a novel PIE development using the digital frequency domain (DFD) technique — FastFLIM, which provides tunable PIE setups and synchronized gating detections, tailored and optimized to specific applications. A few PIE setups by FastFLIM and measurement examples are described. Combined with the sensitivity of Alba and Q2 systems, the PIE allowed us to quantitatively measure the fast dynamics of single molecules.

Long term exposure to arsenic can cause adverse health effects and lead to different levels of disability. The prevalence of arsenical dermatosis is as high as 40% in the Hetao Plain area of Inner Mongolia, but the association between exposure to arsenic in drinking water and the occurrence of disability has not yet been fully examined. The aim of this study was to investigate the prevalence of disability in arsenic-affected villages in Inner Mongolia, China.A cross-sectional study was performed to examine the prevalence of disability. A total of 320 villagers in the age range of 20-39 years were interviewed and examined for disability and arsenical skin lesions. The subjects were classified into a high arsenic group (50 microg L(-1)) and a low arsenic group (50 microg L(-1)). The relationship between levels of arsenic in drinking water and disability was analyzed using multivariate logistic regression models to estimate the odds ratios and 95% confidence intervals.The prevalence of disability was 6.88% in the arsenic affected area of Inner Mongolia and 24.72% in the arsenic group 50 microg L(-1). A strong correlation was found between disability and arsenical skin lesions (OR=86.39, 95%CI: 25.45-293.20).This suggests that the level of arsenic exposure is a major risk factor for disability. Further research is needed to place the results in a wider context and to determine the exact relationship between arsenic exposure and disability.

Purpose: Given the multiple escape mechanisms of tumor cells, immunotherapy targeting tumor-dependent stroma may be an effective cancer treatment strategy. Animal models indicate that inducing immunity to tumor endothelia engenders potent antitumor effects without significant pathology. Recently, the first human tumor endothelial antigen vascular endothelial growth factor receptor-2 (VEGFR-2) recognized by HLA class I–restricted CD8+ T cells has been characterized. In this study, we sought to investigate specific recognition of this molecule by human CD4+ T cells.Experimental Design: To identify HLA-DR–restricted antigenic peptides on VEGFR-2 recognized by CD4+ T cells of healthy donors and cancer patients.Results: Nine candidate VEGFR-2 peptides with high binding probability to six common HLA-DRB1 alleles were synthesized using the SYFPEITHI algorithm. One 15-mer peptide (EKRFVPDGNRISWDS), mapping to the 167-181 region of VEGFR-2, stimulated CD4+ T cells in association with several HLA-DR alleles, including DR4 and DR7. Importantly, the epitope could be naturally processed and presented both by HLA-DR–matched antigen-expressing proliferating endothelial cells and by dendritic cells loaded with the native antigen. Furthermore, circulating VEGFR-2–specific CD4+ T cells were detected in 4 of 10 healthy donors and 12 of 40 cancer patients even after single-round peptide stimulation in short-term culture. Patient's T cells could recognize antigen-expressing proliferating endothelial cells in a HLA-DR–restricted fashion.Conclusion: These findings indicate an important role for the 167-181 region of VEGFR-2 in the stimulation of CD4+ T cell responses to VEGFR-2 protein, and may be instrumental both for the development and monitoring of upcoming antitumor vessel vaccines against different cancers based on VEGFR-2 immunogens.

Forster resonance energy transfer (FRET) is an effective and high resolution method to investigate protein–protein interaction in live or fixed specimens. The FRET technique is increasingly employed to evaluate the molecular mechanisms governing diverse cellular processes such as vesicular transport, signal transduction and the regulation of gene expression. For FRET to occur, protein moieties should be close together within 10 nm, the dipole moment of the fluorophore targeted to the proteins should have an appropriate orientation, and the spectral overlap of the donor emission with the acceptor absorption should be >30 %. FRET can be used to estimate the distance between interacting protein molecules in vivo or in vitro using light microscopy systems. Visible fluorescent proteins (VFPs) have been widely used as a FRET pair in addition to organic dyes. Light microscopy techniques including wide-field, confocal and multiphoton microscopy systems provide spatial information of the interacting proteins with nanometer resolution. For better interpretation and quantitation of the FRET signal the contaminations—also called spectral bleedthrough (SBT)—have to be removed. Another imaging approach, fluorescence lifetime imaging microscopy (FLIM) also provides quantitative information with spatial and temporal details of protein-protein interactions. No algorithm is required here to remove any contamination, as in FLIM-FRET only the change in lifetime value of the donor without and with the acceptor molecules is monitored. The lifetime of the donor decreases at the occurrence of FRET. FLIM is sensitive to the local microenvironment of the molecule but insensitive to the change in fluorophore concentration or excitation intensity. The FLIM-FRET technique is ideal for dark acceptors and the investigation of NADH molecules such as NADH, FAD, Tryptophan, etc. FLIM-FRET techniques provide high temporal resolution of protein-protein interactions in live specimens.

Vaccine strategies that target dendritic cells (DC) in order to elicit immunity against tumors are the subject of intense research. For the induction and maintenance of anti-tumor immunity, CD4+ helper T cells are often required, which need to see appropriate MHC class II-peptide complexes on DC. So far, it remained widely unclear what type of tumor cells can feed the MHC class II processing pathway of DC with what type of antigens. Here, we report that peptide loading onto MHC class II molecules of myeloid DC is facilitated by melanoma cells undergoing necrotic rather than apoptotic cell death. Importantly, the set of MHC class II-associated peptides induced by necrotic tumor cells differed from those found upon engagement of apoptotic tumor cells. This may be due to the fact that only necrotic cells liberated heat shock proteins, which bind tumor-derived peptides and thereby may promote processing by DC. The potential of DC to activate T cells was kinetically controlled through their antigen receptivity: CD4+ T cells were easily stimulated upon encountering antigen early in DC maturation, whereas antigen capture at later maturation stages favored activation of CD8+ T cells. These findings may aid in designing future vaccination strategies and in identifying novel tumor-specific helper T cell antigens.

In the past decade, advances in fluorescence lifetime imaging have extensively applied in the life sciences, from fundamental biological investigations to advanced clinical diagnosis. Fluorescence lifetime imaging microscopy (FLIM) is now routinely used in the biological sciences to monitor dynamic signaling events inside living cells, e.g., Protein-Protein interactions. In this chapter, we describe the calibration of both time-correlated single-photon counting (TCSPC) and frequency domain (FD) FLIM systems and the acquisition and analysis of FLIM-FRET data for investigating Protein-Protein interactions in living cells.

In the past decade, advances in fluorescence lifetime imaging have extensively applied in the life sciences, from fundamental biological investigations to advanced clinical diagnosis. Fluorescence lifetime imaging microscopy (FLIM) is now routinely used in the biological sciences to monitor dynamic signaling events inside living cells, e.g., Protein-Protein interactions. In this chapter, we describe the calibration of both time-correlated single-photon counting (TCSPC) and frequency domain (FD) FLIM systems and the acquisition and analysis of FLIM-FRET data for investigating Protein-Protein interactions in living cells.

Förster Resonance Energy Transfer (FRET) microscopy is a powerful tool used to identify molecular interactions in live or fixed cells using a non-radiative transfer of energy from a donor fluorophore in the excited state to an acceptor fluorophore in close proximity. FRET can be a very sensitive tool to study protein-protein and/or protein-nucleic acids interactions. RNA toxicity is implicated in a number of disorders; especially those associated with expanded repeat sequences, such as myotonic dystrophy. Myotonic dystrophy (DM1) is caused by a (CTG)n repeat expansion in the 3' UTR of the DMPK gene which results in nuclear retention of mutant DMPK transcripts in RNA foci. This results in toxic gain-of-function effects mediated through altered functions of RNA-binding proteins (e.g. MBNL1, hnRNPH, CUGBP1). In this study we demonstrate the potential of a new acceptor photobleaching assay to measure FRET (AP-FRET) between RNA and protein. We chose to focus on the interaction between MBNL1 and mutant DMPK mRNA in cells from DM1 patients due to the strong microscopic evidence of their co-localization. Using this technique we have direct evidence of intracellular interaction between MBNL1 and the DMPK RNA. Furthermore using the AP-FRET assay and MBNL1 mutants, we show that all four zinc-finger motifs in MBNL1 are crucial for MBNL1-RNA foci interactions. The data derived using this new assay provides compelling evidence for the interaction between RNA binding proteins and RNA foci, and mechanistic insights into MBNL1-RNA foci interaction demonstrating the power of AP-FRET in examining RNA-Protein interactions in DM1.

Regulation of products for mucosal application has many points in common with products for other routes but also some that are unique. Identifying regulatory pitfalls is of tremendous importance early in development as this saves time, money, and nerves. There is a huge amount of regulatory advice available from the different players in the field (governments and related organizations) that is partly very general or exceptionally specific. This chapter is meant to provide guidance on what to find, where and who to ask if certain questions arise. It also provides a description of common and known problems differentiated by the three major parts: quality, preclinical, and clinical development. Finally, special key points focusing on pediatric use, use of genetically modified organisms or products classified as advanced therapy medicinal products are discussed.