Excitation Light Intensity Research Articles

Programmable µChopper Device with On-Chip Droplet Mergers for Continuous Assay Calibration.

While droplet-based microfluidics is a powerful technique with transformative applications, most devices are passively operated and thus have limited real-time control over droplet contents. In this report, an automated droplet-based microfluidic device with pneumatic pumps and salt water electrodes was developed to generate and coalesce up to six aqueous-in-oil droplets (2.77 nL each). Custom control software combined six droplets drawn from any of four inlet reservoirs. Using our μChopper method for lock-in fluorescence detection, we first accomplished continuous linear calibration and quantified an unknown sample. Analyte-independent signal drifts and even an abrupt decrease in excitation light intensity were corrected in real-time. The system was then validated with homogeneous insulin immunoassays that showed a nonlinear response. On-chip droplet merging with antibody-oligonucleotide (Ab-oligo) probes, insulin standards, and buffer permitted the real-time calibration and correction of large signal drifts. Full calibrations (LODconc = 2 ng mL−1 = 300 pM; LODamt = 5 amol) required <1 min with merely 13.85 nL of Ab-oligo reagents, giving cost-savings 160-fold over the standard well-plate format while also automating the workflow. This proof-of-concept device—effectively a microfluidic digital-to-analog converter—is readily scalable to more droplets, and it is well-suited for the real-time automation of bioassays that call for expensive reagents.

Optimizing live-cell fluorescence imaging conditions to minimize phototoxicity.

Fluorescence illumination can cause phototoxicity that negatively affects living samples. This study demonstrates that much of the phototoxicity and photobleaching experienced with live-cell fluorescence imaging occurs as a result of 'illumination overhead' (IO). This occurs when a sample is illuminated but fluorescence emission is not being captured by the microscope camera. Several technological advancements have been developed, including fast-switching LED lamps and transistor-transistor logic (TTL) circuits, to diminish phototoxicity caused by IO. These advancements are not standard features on most microscopes and many biologists are unaware of their necessity for live-cell imaging. IO is particularly problematic when imaging rapid processes that require short exposure times. This study presents a workflow to optimize imaging conditions for measuring both slow and dynamic processes while minimizing phototoxicity on any standard microscope. The workflow includes a guide on how to (1) determine the maximum image exposure time for a dynamic process, (2) optimize excitation light intensity and (3) assess cell health with mitochondrial markers.This article has an associated First Person interview with the first author of the paper.

Bright ligand-activatable fluorescent protein for high-quality multicolor live-cell super-resolution microscopy

We introduce UnaG as a green-to-dark photoswitching fluorescent protein capable of high-quality super-resolution imaging with photon numbers equivalent to the brightest photoswitchable red protein. UnaG only fluoresces upon binding of a fluorogenic metabolite, bilirubin, enabling UV-free reversible photoswitching with easily controllable kinetics and low background under Epi illumination. The on- and off-switching rates are controlled by the concentration of the ligand and the excitation light intensity, respectively, where the dissolved oxygen also promotes the off-switching. The photo-oxidation reaction mechanism of bilirubin in UnaG suggests that the lack of ligand-protein covalent bond allows the oxidized ligand to detach from the protein, emptying the binding cavity for rebinding to a fresh ligand molecule. We demonstrate super-resolution single-molecule localization imaging of various subcellular structures genetically encoded with UnaG, which enables facile labeling and simultaneous multicolor imaging of live cells. UnaG has the promise of becoming a default protein for high-performance super-resolution imaging.

基于TPLIF技术的光合荧光参数快速准确测量的激发光强自适应方法

基于TPLIF技术的光合荧光参数快速准确测量的激发光强自适应方法

Protein-Specific, Multicolor and 3D STED Imaging in Cells with DNA-Labeled Antibodies.

Photobleaching is a major challenge in fluorescence microscopy, in particular if high excitation light intensities are used. Signal‐to‐noise and spatial resolution may be compromised, which limits the amount of information that can be extracted from an image. Photobleaching can be bypassed by using exchangeable labels, which transiently bind to and dissociate from a target, thereby replenishing the destroyed labels with intact ones from a reservoir. Here, we demonstrate confocal and STED microscopy with short, fluorophore‐labeled oligonucleotides that transiently bind to complementary oligonucleotides attached to protein‐specific antibodies. The constant exchange of fluorophore labels in DNA‐based STED imaging bypasses photobleaching that occurs with covalent labels. We show that this concept is suitable for targeted, two‐color STED imaging of whole cells.

Protein‐Specific, Multicolor and 3D STED Imaging in Cells with DNA‐Labeled Antibodies

AbstractPhotobleaching is a major challenge in fluorescence microscopy, in particular if high excitation light intensities are used. Signal‐to‐noise and spatial resolution may be compromised, which limits the amount of information that can be extracted from an image. Photobleaching can be bypassed by using exchangeable labels, which transiently bind to and dissociate from a target, thereby replenishing the destroyed labels with intact ones from a reservoir. Here, we demonstrate confocal and STED microscopy with short, fluorophore‐labeled oligonucleotides that transiently bind to complementary oligonucleotides attached to protein‐specific antibodies. The constant exchange of fluorophore labels in DNA‐based STED imaging bypasses photobleaching that occurs with covalent labels. We show that this concept is suitable for targeted, two‐color STED imaging of whole cells.

Aqueous Photon Upconversion by Anionic Acceptors Self-Assembled on Cationic Bilayer Membranes with a Long Triplet Lifetime

Anionic 9,10-diphenylanthracene chromophores electrostatically bound to cationic, chiral bilayer membranes show ordered self-assembly in water. The integrity of the chromophore-accumulated aqueous bilayer membranes is ensured by multiple hydrogen-bond networks introduced in the bilayer, which allowed adaptive accommodation of the guest chromophores at the inner surface of the bilayer while maintaining their cohesive interactions. The regular chromophore alignment in the aqueous assembly is confirmed by differential scanning calorimetry, circular dichroism, and circularly polarized luminescence spectra. Excitonic migration of triplet energy occurs among the chromophores densely organized at the inner surface of the bilayer, which lead to triplet–triplet annihilation-based photon upconversion (TTA-UC). This acceptor-bilayer self-assemblies show a notably long triplet lifetime of 8.0 ms, which allows TTA-UC at sufficiently low excitation light intensity. These results demonstrate the usefulness of the simple electrostatic accumulation approach for TTA-UC chromophores where the suitable molecular design of the TTA-UC chromophore-integrated bilayer membranes plays a key role.

Influence of Exciting-Light Intensity on the Concentration Dependence of Organic Molecules Luminescence in Glassy Matrices*

Triplet-level population distributions of organic molecules in glassy matrices were studied by computer simulation for various exciting-light intensities and compound concentrations. The dependence of the triplet-level population distribution on these variables was found. A consequence of the established relationship was that the exciting-light intensity affected the concentration dependence of the luminescence intensity where the population change of the molecular ground state resulting from their transition into the triplet state could not be neglected.

UV light irradiation enhanced gas sensor selectivity of NO2 and SO2 using rGO functionalized with hollow SnO2 nanofibers

Herein, to enhance selective detection of NO2 and SO2 using a single device, an ultraviolet (UV) light activated gas sensor has been reported based on reduced graphene oxide (rGO) functionalized with hollow SnO2 nanofibers (NFs) at room temperature. The porous hollow SnO2 NFs are synthesized using electrospinning and calcination treatment, then different contents of SnO2 are introduced to rGO for preparation of rGO/SnO2 nanocomposites by facile magnetic stirring and ultrasonic treatment. The rGO/SnO2 samples reveal obvious sensing response, great reversibility and good humidity resistance to target gases at ppm level. Under different intensities of UV light illumination, gas sensing properties of rGO/SnO2 are studied to explore the photoexcited sensing behaviors. The greatly enhanced selective detection to NO2 (102%) and SO2 (11%) is achieved with a response ratio of 9.3 by using UV light illumination. The results show sensor response and selectivity can be improved using appropriate intensity of excitation light, demonstrating significant modulation effect of UV light assistance on gas detection. The mechanism may be attributed to light motivated electron-hole pairs due to built-in electric fields under UV light illumination, which can be captured by target gases and lead to UV controlled gas sensing performance.

PyPhotometry: Open source Python based hardware and software for fiber photometry data acquisition

Fiber photometry is the process of recording bulk neural activity by measuring fluorescence changes in activity sensitive indicators such as GCaMP through an optical fiber. We present a system of open source hardware and software for fiber photometry data acquisition consisting of a compact, low cost, data acquisition board built around the Micropython microcontroller, and a cross platform graphical user interface (GUI) for controlling acquisition and visualising signals. The system can acquire two analog and two digital signals, and control two external LEDs via built in LED drivers. Time-division multiplexed illumination allows independent readout of fluorescence evoked by different excitation wavelengths from a single photoreceiver signal. Validation experiments indicate this approach offers better signal to noise for a given average excitation light intensity than sinusoidally-modulated illumination. pyPhotometry is substantially cheaper than commercial hardware filling the same role, and we anticipate, as an open source and comparatively simple tool, it will be easily adaptable and therefore of broad interest to a wide range of users.

Photoinduced photoluminescence enhancement in self-assembled clusters of formamidinium lead bromide perovskite nanocrystals.

Nanocrystals of formamidinium lead bromide perovskite (FAPbBr3) self-assemble into clusters in powder and film samples and provide a prolonged photoluminescence lifetime, which is attributed to the diffusion of charge carriers through interparticle states formed among nanocrystals. Interestingly, the photoluminescence lifetime decreases and the emission intensity increases for the clusters, which is with the increase in the intensity of excitation light. By doping the nanocrystal clusters with C60, we successfully harvested the photogenerated charge carriers. Nonetheless, at high intensities of excitation, the rate of radiative recombination becomes comparable to that of the electron transfer to C60. Thus, the optimum rate of electron transfer to C60 is accomplished by minimally exciting the self-assembled nanocrystals.

Photoinduced Mn doping in cesium lead halide perovskite nanocrystals.

We report the photoinduced post-synthesis method of Mn doping in colloidal perovskite nanocrystals, which can produce Mn-doped CsPbX3 (X = Cl, Br) nanocrystals with preserved size and anisotropic morphology. Photoinduced Mn doping occurs through cation exchange driven by the facile photoinduced halide exchange in dihalomethane (CH2X2, X = Cl, Br) solvent taking advantage of in situ photogeneration of halide ions from the solvent molecules. In the presence of a small amount of Mn acetate dissolved in solvent at sub-micromolar concentration, photoexcitation of the nanocrystals above the bandgap initiates the simultaneous anion and cation exchange. Under the condition of self-anion exchange, the resulting product is only the cation (Mn) doping in the nanocrystal host without changing halide composition, where the extent of doping can be controlled by excitation light intensity. The mild nature of the photoinduced doping also preserves the anisotropic morphology of the nanocrystals. The photoinduced Mn-doping method could be further expanded to other cations providing a versatile means of creating various cation-doped perovskite nanocrystals that are difficult to produce by other means.

Triarylmethane Fluorophores Resistant to Oxidative Photobluing.

Spectral stability of small-molecule fluorescent probes is required for correct interpretation and reproducibility of multicolor fluorescence imaging data, in particular under high (de)excitation light intensities of super-resolution imaging or in single-molecule applications. We propose a synthetic approach to a series of spectrally stable rhodamine fluorophores based on sequential Ru- and Cu-catalyzed transformations, evaluate their stability against photobleaching and photoconversion in the context of other fluorophores using chemometric analysis, and demonstrate chemical reactivity of fluorophore photoproducts. The substitution patterns providing the photoconversion-resistant triarylmethane fluorophores have been identified, and the applicability of nonbluing labels in live-cell STED nanoscopy is demonstrated.

Amplified and Multicolor Emission from Films and Interfacial Layers of Lead Halide Perovskite Nanocrystals

Lead halide perovskites are promising for energy harvesting and lighting applications. We show amplified emission from films of organic–inorganic or all-inorganic lead halide perovskite nanocrystals. Also, multicolor emission is detected from interfacial layers of chloride and bromide perovskites. While amplified emission originates from spatial confinement of manifold charge carriers in films, multicolor emission emanates from interfacial halogen-exchanged layers. In a film, the amplified emission is averaged over a large number of nanocrystals, which prevents us from detecting any spectral narrowing. Conversely, amplified spontaneous emission (ASE) and spectral narrowing are detected from an isolated perovskite microcrystal. Interestingly, under low-intensity excitation, a perovskite nanocrystal film shows unusually delayed emission, which becomes extremely fast and spatially confined at higher intensities of excitation light. By precisely detecting photoluminescence from an irradiated area and outside,...

Precise Control of Localized Surface Plasmon Wavelengths Is Needed for Effective Enhancement of Triplet–Triplet Annihilation-Based Upconversion Emission

In this study, we demonstrate that the localized surface plasmon (LSP) resonance of metal nanoparticles, depending strictly on the generating wavelength of LSP resonance, can have both beneficial enhancement and harmful quenching effects on a triplet–triplet annihilation-based upconversion (TTA-UC) emission. When the LSP resonance band of anisotropic silver nanoprisms spectrally overlapped with the photoexcitation wavelength of a sensitizer and the fluorescence of an emitter, an increase in the photoexcitation efficiency and an acceleration of the radiative decay rate were respectively induced, resulting in an effective enhancement in the TTA-UC emission. Furthermore, the overlapping with the photoexcitation wavelength led to a significant decrease (93%) in the threshold light excitation intensity, which greatly enhances the figure-of-merit in TTA-UC systems. However, when the LSP resonance band overlapped with the phosphorescence band of the sensitizer, the TTA-UC emission was extremely quenched, accompa...

Light-Controlled in Situ Bidirectional Tuning and Monitoring of Gold Nanorod Plasmon via Oxidative Etching with FeCl3

We report on light-controlled in situ bidirectional tuning of longitudinal surface plasmon resonance (LSPR) of single gold nanorods via oxidative etching with ferric chloride. By removing the surfactant layer from the surface of a gold nanorod, we demonstrate that the etching happens only in the presence of an excitation laser, and the etching rate and directionality can be controlled by the intensity of excitation light. At a low excitation power, a blue shift of a nanorod’s LSPR of up to 50 nm was observed, which indicates preferential etching from its tips. Whereas at a high power, we see a red shift of the nanorod’s LSPR of up to 140 nm indicating etching from sides. These results present a new approach for in situ finer adjustments of a selected nanorod’s plasmon resonance.

Phototoxicity of flavoprotein miniSOG induced by bioluminescence resonance energy transfer in genetically encoded system NanoLuc-miniSOG is comparable with its LED-excited phototoxicity

Photodynamic therapy (PDT) is a clinical, minimally invasive method for destroying cancer cells in the presence of a photosensitizer, oxygen, and a light source. The main obstacle for the PDT treatment of deep tumors is a strong reduction of the excitation light intensity as a result of its refraction, reflection, and absorption by biological tissues. Internal light sources based on bioluminescence resonance energy transfer can be a solution of this problem. Here we show that luciferase NanoLuc being expressed as a fusion protein with phototoxic flavoprotein miniSOG in cancer cells in the presence of furimazine (highly specific NanoLuc substrate) induces a photodynamic effect of miniSOG comparable with its LED-excited (Light Emitting Diode) phototoxicity. Luminescence systems based on furimazine and hybrid protein NanoLuc-miniSOG targeted to mitochondria or cellular membranes possess the similar energy transfer efficiencies and similar BRET-induced cytotoxic effects on cancer cells, though the mechanisms of BRET-induced cell death are different. As the main components of the proposed system for BRET-mediated PDT are genetically encoded (luciferase and phototoxic protein), this system can potentially be delivered to any site in the organism and thus may be considered as a promising approach for simultaneous delivery of light source and photosensitizer in deep-lying tumors and metastasis anywhere in the body.

Stepwise photochromism of bisnaphthopyrans exhibiting an excitation intensity-dependent color change.

Non-linear photoresponses against excitation light intensity are important for the development of attractive photofunctional materials exhibiting high spatial selective photoswitching that is not affected by weak background light. Biphotochromic systems composed of two fast photochromic units have the potential to show a stepwise two-photon absorption process in which the optical properties can be non-linearly controlled by changing the excitation light conditions. Herein, we designed and synthesized novel bisnaphthopyran derivatives containing fast photoswitchable naphthopyran units. The bisnaphthopyran derivatives show a stepwise two-photon-induced photochromic reaction upon UV light irradiation accompanied by a drastic color change due to a large change in the molecular structure between the one-photon product and the two-photon product. Consequently, the color of the bisnaphthopyran derivatives can be non-linearly controlled by changing the excitation intensity. This characteristic photochromic property of the biphotochromic system provides important insight into advanced photoresponsive materials.

Investigation of Electrical Parameters of Amorphous–Crystalline Silicon Heterojunction Solar Cells: Correlations Between Carrier Dynamics and S-Shape of Current Density–Voltage Curve

We have analyzed a-Si:H(p)/a-Si:H(i)/c-Si(n) heterojunction silicon solar cell having the S-shaped current density–voltage characteristics with a low fill factor and open-circuit voltage, using quantum efficiency (QE) characterization technique under forward/reverse voltage and different light (blue, infrared, and white) bias conditions. The curvature of S-shape is sensitive to excitation light intensities because of modification in junction barrier potential (variation in quasi-Fermi levels splitting). With forward-bias voltage alone near/above S-shaped region, cell's QE is uniformly reduced because of reduction in junction field and dominance of barrier for collection of holes. However, with blue and white light at bias voltages close to S-shaped characteristics, a uniform improvement of QE in broad wavelength region is observed because of defects saturation at the junction interface and photoconductivity in the a-Si layers. With white light and voltage bias, cell's QE is anomalously improved and it has even crossed the QE response at no voltage/light bias conditions in the blue region because of defects saturation in a-Si:H layers, whereas under infrared light and voltage bias conditions defect saturation is not displayed in the QE because of carrier generation in a deeper region of the cell after crossing unabsorbed photons front region.

Morphological Contributions to Interfacial Charge Trapping and Nongeminate Recombination in Polymer Solar Cells Revealed by UV Light Soaking.

Nongeminate charge recombination occurs over a broad range of time scales in polymer solar cells and represents a serious loss channel for the performance and lifetime of devices. Multiple factors influence this process, including changes in morphology and formation of permanent defects, but individual contributions are often difficult to resolve from conventional experiments. We use intensity modulated photocurrent/photovoltage spectroscopy (IMPS/IMVS) to investigate nongeminate charge recombination in blends of poly[2,6-(4,4-bis-(2-ethylhexyl)-4 H-cyclopenta [2,1- b;3,4- b']dithiophene)- alt-4,7(2,1,3-benzothiadiazole)] (PCPDTBT) and [6,6]-phenyl-C61-butyric acid methyl ester (PCBM) solar cells. PCPDTBT/PCBM devices are exposed to varying doses of UV light resonant with PCBM to induce small perturbations in the thin film morphology, namely local heating. IMPS/IMVS sweeps display signatures unique to degradation, that is, photocurrent and photovoltage leading the excitation light modulation appearing as positive phase shifts or 1st quadrant features in Bode and Nyquist representations, respectively. We assign this component to interface charging at purified PCPDTBT/PCBM phase boundaries that trap mobile charges and facilitate nongeminate recombination. Time- and frequency-domain drift-diffusion simulations are then used to model the perturbed photocurrent responses that show good agreement with experiments. Trap occupancies and their impact of photocurrent production are investigated using variable background (dc) excitation light intensities revealing increases of the 1st quadrant component in devices irradiated for longer times. No evidence of chemical degradation was observed from molecular spectroscopy and imaging experiments, and we conclude that morphological changes are chiefly responsible for larger nongeminate charge recombination yields as devices age. Lastly, we propose that the 1st quadrant IMPS/IMVS is a universal signature of morphology-related degradation, although its relative contribution may vary between material systems.