Repetitive Illumination Research Articles

Multispectral confocal 3D imaging of intact healthy and tumor tissue using mLSR-3D.

Revealing the 3D composition of intact tissue specimens is essential for understanding cell and organ biology in health and disease. State-of-the-art 3D microscopy techniques aim to capture tissue volumes on an ever-increasing scale, while also retaining sufficient resolution for single-cell analysis. Furthermore, spatial profiling through multi-marker imaging is fast developing, providing more context and better distinction between cell types. Following these lines of technological advance, we here present a protocol based on FUnGI (fructose, urea and glycerol clearing solution for imaging) optical clearing of tissue before multispectral large-scale single-cell resolution 3D (mLSR-3D) imaging, which implements 'on-the-fly' linear unmixing of up to eight fluorophores during a single acquisition. Our protocol removes the need for repetitive illumination, thereby allowing larger volumes to be scanned with better image quality in less time, also reducing photo-bleaching and file size. To aid in the design of multiplex antibody panels, we provide a fast and manageable intensity equalization assay with automated analysis to design a combination of markers with balanced intensities suitable for mLSR-3D. We demonstrate effective mLSR-3D imaging of various tissues, including patient-derived organoids and xenografted tumors, and, furthermore, describe an optimized workflow for mLSR-3D imaging of formalin-fixed paraffin-embedded samples. Finally, we provide essential steps for 3D image data processing, including shading correction that does not require pre-acquired shading references and 3D inhomogeneity correction to correct fluorescence artefacts often afflicting 3D datasets. Together, this provides a one-week protocol for eight-fluorescent-marker 3D visualization and exploration of intact tissue of various origins at single-cell resolution.

Annealing effect of pentacene and ultrathin metal anode buffer on the performance of small-molecule organic solar cells

The annealing effect of thin pentacene and ultrathin gold/silver anode buffer layers on both the initial performance and durability against repetitive illumination stress in small-molecule organic solar cells (OSCs) was experimentally investigated. The OSC had a structure of indium tin oxide (ITO, anode)/anode buffer/copper phthalocyanine (CuPc, donor)/fullerene (acceptor)/bathocuproine (cathode buffer)/Ag (cathode). OSCs with a 3-nm-thick pentacene anode buffer layer provided a 15% increase in the power conversion efficiency compared to OSCs with no pentacene layer. OSCs with an ultrathin 0.3-nm-thick Au anode buffer layer provided an increase of 63% in durability compared to OSCs without the Au layer. OSCs with the Au layer did not show apparent leakage current, while those with the Ag layer did. OSCs with a combinational anode buffer of the pentacene layer/Au layer were mainly examined. The spatial distribution of Au atoms on pentacene was uniform even after annealing, which helped to improve the quality of CuPc crystals. The ultrathin Au layer brought both a large absorption of ultraviolet (UV) light and highly dense nucleation sites for overlaid CuPc film formation. These were possible reasons for durability improvement. The Au layer thickness of 0.3 nm on pentacene was optimal to achieve the condition with both less attenuation of visible light necessary for electric power generation and larger absorption of UV light causing degradation. Annealing treatment before CuPc evaporation brought both preferable initial performance and durability for OSCs with a combinational structure of the ITO/pentacene layer/Au layer. An appropriate region of annealing temperature had, however, a small window around 100°C.

A Geometric Correspondence Feature Based-Mismatch Removal in Vision Based-Mapping and Navigation

Images with large-area repetitive texture, significant viewpoint, and illumination changes as well as occlusions often induce high-percentage keypoint mismatches, affecting the performance of vision-based mapping and navigation. Traditional methods for mismatch elimination tend to fail when the percentage of mismatches is high. In order to remove mismatches effectively, a new geometry-based approach is proposed in this paper, where Geometric Correspondence Feature (<small>GCF</small>) is used to represent the tentative correspondence. Based on the clustering property of <small>GCFs</small> from correct matches, a new clustering algorithm is developed to identify the cluster formed by the correct matches. With the defined quality factor calculated from the identified cluster, a Progressive Sample Consensus (<small>PROSAC</small>) process integrated with hyperplane-model is employed to further eliminate mismatches. Extensive experiments based on both simulated and real images in indoor and outdoor environments have demonstrated that the proposed approach can significantly improve the performance of mismatch elimination in the presence of high-percentage mismatches.

Stability in organic solar cells with Ag/pentacene anode buffer under repetitive illumination

The degradation phenomenon in small-molecule organic solar cells (OSCs) with a structure of indium-tin oxide (ITO, anode)/anode buffer/copper phthalocyanine (CuPc, donor)/fullerene (acceptor)/bathocuproine (cathode buffer)/Ag (cathode) was experimentally investigated. Two kinds of anode buffer layers, a 2-nm-thick pentacene layer and a 0.5-nm-thick Ag layer, were examined. The devices were subjected to repetitive illumination stress through the transparent ITO electrode in air. The ultra-thin Ag layer enhanced the ability of the devices to withstand the illumination stress. A thinner Ag layer resulted in better device performance. Resonant light absorption in the nanometer-scale Ag clusters probably accumulates trapped electrons near the ITO/CuPc interface, shrinks the depletion layer, enhances tunneling injection of holes, and suppresses the S-shaped kink during the illumination. The thin pentacene layer provides good ohmic contact between the anode and the CuPc layer, resulting in a larger short-circuit current. OSCs fabricated with both Ag and pentacene anode buffer layers exhibited only a 10% decrease in power conversion efficiency at 60 repetitions, whereas OSCs without an anode buffer layer exhibited a decrease of approximately 45%. The large decrease in efficiency was dominated by the decrease in the fill factor.

Degradation in organic solar cells under illumination and electrical stresses in air

The degradation phenomenon in small molecular organic solar cells with an indium tin oxide (anode)/copper phthalocyanine (donor)/fullerene (acceptor)/bathocuproine (buffer)/Ag (cathode) structure is experimentally investigated. The effect of three kinds of stress, that is, air exposure, repetitive illumination, and repetitive electrical stress, on device performance is examined. Both air exposure and repetitive illumination deteriorate the device performance appreciably. The decrease in power conversion efficiency is dominated by the decrease in the fill factor. Repetitive electrical stress does not cause substantial device degradation. The degree of degradation under a combination of stresses from repetitive illumination and air exposure is lower than that under illumination stress alone. Furthermore, there is a possibility that this combination of stresses induces some passivation against further air exposure stress.

In vitro experimental study of novel porphyrin-typed photosensitizer photodynamically treating hepatic carcinoma

Objective To study the photodynamic therapy (PDT) mediated by a novel porphyrin-typed photosensitizer on human hepatic carcinoma HepG2 cell and the mechanisms.Methods Experiments were derided into four groups:control group,PDT group,photosensitizer group and photosensitizer+PDT group.The photostability of novel photosensitizer upon repetitive illumination was evaluated by bleaching method,and cell survival rate was determined by MTT assay.Cellular uptake of novel photosensitizer was measured with luminescence spectrometer,and cellular localization ofphotosensitizer was observed by laser scanning confocal microscopy (LSCM).Furthermore,apoptotic cell was detected with Hoechst 333342 staining.Results Novel photosensitizer was stable after repetitive light irradiation,and PDT or photosensitizer alone showed no dark cytotoxicity toward HepG2 cell (P＞0.05),but intense killing was observed in photosensitizer+PDT group (P＜0.05).The IC50 is 1.21 μmol/L.Cellular uptake of novel photosensitizer was concentration-dependent and the highest uptake is at concentration of 12.5 μmol/L.Novel photosensitizer localizes in lysosomes of HepG2 cell,and the death mode of HepG2 cell was mainly apoptosis.Conclusions Novel photosensitizer exerts profound cytotoxic effects on HepG2 cell mainly through the initiation of secondary cell apoptosis by lysosome destruction. Key words: Novel photosensitizer; HepG2 cell; Photosensitizer uptake; Subcellular localization; Apoptosis

Repetitive short-pulse light mainly inactivates photosystem I in sunflower leaves.

Under field conditions, the leaves of plants are exposed to fluctuating light, as observed in sunfleck. The duration and frequency of sunfleck, which is caused by the canopy being blown by the wind, are in the ranges from 0.2 to 50 s, and from 0.004 to 1 Hz, respectively. Furthermore, >60% of the sunfleck duration ranges from 0.2 to 0.8 s. In the present research, we analyzed the effects of repetitive illumination by short-pulse (SP) light of sunflower leaves on the photosynthetic electron flow. The duration of SP light was set in the range from 10 to 300 ms. We found that repetitive illumination with SP light did not induce the oxidation of P700 in PSI, and mainly inactivated PSI. Increases in the intensity, duration and frequency of SP light enhanced PSI photoinhibition. PSI photoinhibition required the presence of O2. The inactivation of PSI suppressed the net CO2 assimilation. On the other hand, the increase in the oxidized state of P700 suppressed PSI inactivation. That is, PSI with a reduced reaction center would produce reactive oxygen species (ROS) by SP light, leading to PSI photodamage. This mechanism probably explains the PSI photodamage induced by constant light.

A new photodynamic therapy drug toward gastric cancer MGC803 cell

To investigate the treatment efficiency of a new photodynamic therapeutic(PDT) drug synthesized by our laboratory toward MGC803 cells and related mechanisms. Bleaching method was used to evaluate the photostability of drug upon repetitive illumination. MTT assay was used to determine the ability of new drug killing MGC803 cells after PDT. Laser scanning confocal microscopy (LSCM) was applied to investigate the subcellular localization of drug in MGC803 cells (mitochondria and/or lysosomes). Hoechst staining and flow cytometry(Annexin V/PI double-staining) were performed to detect the death mode of MGC803 cells after PDT. This new PDT drug had good stability to light irradiation after repetitive illumination. MTT assay showed no cytotoxicity towards MGC803 cells only by drug or only by irradiation(P>0.05), but intense lethal effect was observed with drug and light combination(P<0.05). The phototoxicity of medicine increased with the elevation of concentration, the LD50 was 1.74 μmol/L, and reaching plateau at the concentration of 3.12 μmol/L, even increasing the concentration. LSCM found that drug localized in lysosomes of MGC803 cells. Hoechst staining showed that the death mode of cells was mainly necrosis and Annexin V/PI double-staining proved this result further. This new PDT drug is an effective PDT sensitizer for MGC803 cells and the death mode of cells is mainly necrosis.

Sub–damage–threshold femtosecond laser ablation from crystalline Si: surface nanostructures and phase transformation

Surface structures and structural transformations are investigated upon femtosecond laser ablation (800 nm, 120 fs) from crystalline silicon (100) targets placed under ultra-high vacuum. After repetitive illumination with several thousand laser pulses at intensities below the single shot damage threshold, at normal incidence, the crater morphology indicates the development of periodic structures at the crater bottom, with the orientation depending on the laser beam polarization. Periods of 200 nm and 600–700 nm, respectively, are shorter than the laser wavelength and appear as a result of surface instability. The ablation dynamics monitored by time-of-flight mass spectrometry shows the emission of positive silicon ions and clusters with kinetic energies of about 7 eV. Raman spectroscopy reveals phase transformations in the irradiated spot from Si-I to the polymorphs Si-III, Si-IV, Si-XII, and amorphous silicon as well as a stable, uncommon phase of hexagonal Si-wurzite.

Rear-surface laser damage on 355-nm silica optics owing to Fresnel diffraction on front-surface contamination particles

Light intensity modulations caused by opaque obstacles (e.g., dust) on silica lenses in high-power lasers often enhance the potential for laser-induced damage. To study this effect, particles (10-250 mum) with various shapes were sputter deposited on the input surface and irradiated with a 3-ns laser beam at 355 nm. Although a clean silica surface damages at fluences above 15 J/cm(2), a surface contaminated with particles can damage below 11.5 J/cm(2). A pattern that conforms to the shape of the input surface particle is printed on the output surface. Repetitive illumination resulted in catastrophic drilling of the optic. The damage pattern correlated with an interference image of the particle before irradiation. The image shows that the incident beam undergoes phase (and amplitude) modulations after it passes around the particle. We modeled the experiments by calculating the light intensity distribution behind an obscuration by use of Fresnel diffraction theory. The comparison between calculated light intensity distribution and the output surface damage pattern showed good agreement. The model was then used to predict the increased damage vulnerability that results from intensity modulations as a function of particle size, shape, and lens thickness. The predictions provide the basis for optics cleanliness specifications on the National Ignition Facility to reduce the likelihood of optical damage.

Calcium ion, an intracellular messenger of light adaptation, also participates in excitation of Limulus photoreceptors.

Photoreceptor cells of Limulus ventral eyes were bathed in artificial sea water (ASW) that contained 10 mM-EGTA and no added Ca2+ (EGTA-ASW). Test flashes elicited responses that increased to a maximum size within 10 min in EGTA-ASW but did not change further when dark-adapted cells were bathed for an additional 35 min in this solution. Light responses progressively declined from this maximum size if the cells were repetitively illuminated in EGTA-ASW. In this state of reduced responsiveness, response amplitudes were further reduced by intracellular ionophoretic injection of EGTA; response amplitudes were increased by intracellular ionophoretic injection of Ca2+. Both of these findings are opposite to what is normally observed for cells bathed in ASW. Also, after repetitive illumination in EGTA-ASW, both the slope of the response versus intensity relationship became steeper and light responses often had a delayed increase in amplitude. The light responses and the response versus intensity relation returned to normal when the bathing medium was changed back to ASW containing 10 mM-Ca2+. The light-induced rise in luminescence recorded from photoreceptors injected with the photoprotein aequorin (the 'aequorin response') declined by at most 50% after dark-adapted photoreceptors were bathed in EGTA-ASW for 45 min. However, the aequorin response progressively declined by 98% if cells were repetitively illuminated while bathed in EGTA-ASW. The total intracellular Ca content of whole end-organs was measured by atomic absorption spectroscopy. Total intracellular Ca content did not change significantly while photoreceptors were bathed in EGTA-ASW even after repetitive illumination. We suggest that cytosolic Ca2+ is required by one or more steps in the mechanisms that link rhodopsin isomerization to both (i) an increase in the conductance of the cell membrane to Na+ and (ii) a release of Ca2+ from a light-labile store.

A Study of the Environmental Impact of an Intense Flashing Airport Lighting System

We report herein what we believe to be the first effort to evaluate the environmental impact of a relatively bright flashing illumination system. The system in question is the medium-intensity approach lighting system with runway alignment indicator lights (MALSR) that is the optical guidance system of choice in most precision landing systems today. We have examined the question of whether the repetitive flashing illumination is likely to induce abnormal perceptual or motor effects or cause serious distraction and interference with the normal activities of people who may be inadvertently illuminated by the MALSR. Included are an evaluation of the illumination of this particular system and the psychophysical response to this kind of flickering illumination, a discussion of our criteria for judging the impact of intrusive illumination, and finally, direct measurements of the very high immunity to extraneous distraction for motivated human operators attempting to do complex visual tasks with a field of view ...

Picosecond Laser-Induced Surface Transformations in Solids

ABSTRACTWe report the results of a study of surface transformations in semiconductors (Si, GaAs) and metals (Cu) produced by 100 ps long pulses from a Nd:YAG laser system. We present and outline the conclusions of our model for growth of spontaneous periodic surface structures or ripples often associated with laser damage and annealing. The effect of repetitive subthreshold illumination is also examined. Theoretical analysis is presented to support time-resolved experiments performed with two excitation pulses at different wavelengths.

Photoinactivation of the methotrexate transport system of L1210 cells by 8-azidoadenosin 5'-monophosphate.

Methotrexate transport in L1210 cells is mediated by a carrier protein that can bind organic and inorganic phosphate compounds in addition to the various folate substrates. The photoaffinity labeling agent, 8-azidoadenosine 5'-monophosphate (8-azido-AMP), also interactis (Ki = 140 microM) with the receptor site for this transport system, and upon irradiation with ultraviolet light, irreversibly inhibits methotrexate uptake. Protection against this inactivation is afforded by either a substrate (methotrexate) or a competitive inhibitor (inorganic phosphate). The light-induced reaction proceeds rapidly (t1/2 = 2 min at 23 degrees C under the conditions described) and produces half-maximal reduction in the transport rate when the 8-azido-AMP concentration is 65 microM. complete photoinactivation of methotrexate transport could not be obtained from a single exposure to 8-azido-AMP (up to 1.0 mM), but it could be achieved by the repetitive illumination of cells in a fresh medium. The phosphate and folate/adenine transport systems of L1210 cells are not affected by irradiation in the presence of 8-azido-AMP.

Time-of-Flight Electron Velocity Spectrometer

A velocity selector is described which is designed for measurement of the total collision cross section of electrons in the energy region below 1 V. The system determines the distributions of times of flight of single electrons over a measured path at selected pressures of a specimen gas. The electrons are generated by repetitive illumination of a photoemitter by a spark gap, pass over a shielded drift path, enter an accelerating field, and impinge on an electron multiplier. Electronic equipment and data reduction for analysis of time-of-flight and amplitude distributions of electron counts are described; data illustrative of performance are presented, including typical transmission measurements in helium and argon which extend to 150 mV.

PRIMATE RETINAL RESPONSES: SLOW CHANGES DURING REPETITIVE STIMULATION WITH LIGHT.

Sudden, repetitive illumination of the dark-adapted monkey eye produces transient changes in the electroretinogram and transocular potential which can last an hour or longer.