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Gao, Melisa, George Lewis, Gordon M. Turner, Antoine Soubret, and Vasilis Ntziachristos. "Effects of background fluorescence in fluorescence molecular tomography." Applied Optics 44, no. 26 (September 10, 2005): 5468. http://dx.doi.org/10.1364/ao.44.005468.
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Sun, Yulong, and Avi Chakrabartty. "Cost-effective elimination of lipofuscin fluorescence from formalin-fixed brain tissue by white phosphor light emitting diode array." Biochemistry and Cell Biology 94, no. 6 (December 2016): 545–50. http://dx.doi.org/10.1139/bcb-2016-0125.
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Autofluorescence of aldehyde-fixed tissues greatly hinders fluorescence microscopy. In particular, lipofuscin, an autofluorescent component of aged brain tissue, complicates fluorescence imaging of tissue in neurodegenerative diseases. Background and lipofuscin fluorescence can be reduced by greater than 90% through photobleaching using white phosphor light emitting diode arrays prior to treatment with fluorescent probes. We compared the effect of photobleaching versus established chemical quenchers on the quality of fluorescent staining in formalin-fixed brain tissue of frontotemporal dementia with tau-positive inclusions. Unlike chemical quenchers, which reduced fluorescent probe signals as well as background, photobleaching treatment had no effect on probe fluorescence intensity while it effectively reduced background and lipofuscin fluorescence. The advantages and versatility of photobleaching over established methods are discussed.
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Kwon, Sunkuk, and Eva M. Sevick-Muraca. "Effects of Depilation-Induced Skin Pigmentation and Diet-Induced Fluorescence on In Vivo Fluorescence Imaging." Contrast Media & Molecular Imaging 2017 (2017): 1–7. http://dx.doi.org/10.1155/2017/7659242.
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Near-infrared fluorescence imaging (NIRFI) and far-red fluorescence imaging (FRFI) were used to investigate effects of depilation-induced skin pigmentation and diet-induced background fluorescence on fluorescent signal amplitude and lymphatic contraction frequency in C57BL6 mice. Far-red fluorescent signal amplitude, but not frequency, was affected by diet-induced fluorescence, which was removed by feeding the mice an alfalfa-free diet, and skin pigmentation further impacted the amplitude measurement. NIRFI showed minimal background fluorescence; however, skin pigmentation reduced the amplitude of fluorescent signal changes. Therefore, these effects should be taken into account when imaging mice with different states of skin pigmentation and diet-induced background fluorescence in vivo.
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Wang, Nam Sun, and Michael B. Simmons. "Effect of background fluorophores on the NADH fluorescence probe signal." Biotechnology Techniques 5, no. 4 (July 1991): 241–46. http://dx.doi.org/10.1007/bf02438655.
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Kono, Luna, Yuma Nakagawa, Ayako Fujimoto, Ryo Nishimura, Yohei Hattori, Toshiki Mutai, Nobuhiro Yasuda, et al. "Aggregation-induced emission effect on turn-off fluorescent switching of a photochromic diarylethene." Beilstein Journal of Organic Chemistry 15 (September 20, 2019): 2204–12. http://dx.doi.org/10.3762/bjoc.15.217.
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Background: Diarylethenes are well-known photochromic compounds, which undergo cyclization and cycloreversion reactions between open- and closed-ring isomers. Recently, diarylethene derivatives with photoswitchable fluorescent properties were prepared. They are applicable for fluorescence imaging including bio-imaging. On the other hand, a new system called “excited state intramolecular proton transfer (ESIPT)” is reported. In the system, absorption and emission bands are largely separated due to the proton transfer, hence it showed strong fluorescence even in the crystalline state. We aimed to construct the photochromic system incorporating the ESIPT mechanism. Results: A diarylethene incorporating a fluorescent moiety that exhibit ESIPT behavior was prepared. The ESIPT is one of the examples which express the mechanisms of aggregation-induced emission (AIE). This compound emits orange fluorescence with a large Stokes shift derived from ESIPT in aprotic solvents such as THF or hexane, while it exhibits only a photochromic reaction in protic solvents such as methanol. In addition, it shows turn-off type fluorescence switching in an aprotic solvent and in crystals. The fluorescence is quenched as the content of closed-ring isomers increases upon UV light irradiation. Conclusions: A diarylethene containing an ESIPT functional group was prepared. It showed fluorescent turn-off behavior during photochromism in aprotic solvents as well as in crystalline state upon UV light irradiation. Furthermore, it showed AIE in THF/water mixtures with blue-shift of the emission.
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Rosati, Anna, Luigi Candussio, Enrico Crivellato, Fiora Bartoli Klugmann, Tullio Giraldi, Daniela Damiani, Angela Michelutti, and Giuliana Decorti. "Bodipy-FL-Verapamil: A Fluorescent Probe for the Study of Multidrug Resistance Proteins." Analytical Cellular Pathology 26, no. 1-2 (January 1, 2004): 3–11. http://dx.doi.org/10.1155/2004/576173.
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Most of the substances used as fluorescent probes to study drug transport and the effect of efflux blockers in multidrug resistant cells have many drawbacks, such as toxicity, unspecific background, accumulation in mitochondria. New fluorescent compounds, among which Bodipy‐FL‐verapamil (BV), have been therefore proposed as more useful tools. The uptake of BV has been evaluated by cytofluorimetry and fluorescence microscopy using cell lines that overexpress P‐glycoprotein (P388/ADR and LLC‐PK1/ADR) or MRP (multidrug resistance‐related protein) (PANC‐1) and clinical specimens from patients. The effect of specific inhibitors for P‐glycoprotein (verapamil and vinblastine) or MRP (MK571 and probenecid) has been also studied. BV intracellular concentrations were significantly lower in the two P‐glycoprotein overexpressing cell lines in comparison with the parental lines. In addition, verapamil and vinblastine increased the intracellular concentrations of the dye; MK571 and probenecid, two MRP inhibitors, increased BV levels in PANC‐1 cells, that express this protein. These findings were confirmed in clinical specimens from patients. Fluorescence microscopy revealed a faint fluorescence emission in P‐glycoprotein or MRP expressing cell lines; however, treatment with specific inhibitors significantly increased the fluorescence. BV is a useful tool for studying multidrug resistance proteins with different techniques such as cytofluorimetry and fluorescence microscopy, but does not discriminate between P‐glycoprotein and MRP. In comparison with other classic fluorescent probes, the assay with this dye is extremely rapid, simple, not toxic for cells, devoid of fluorescent background, and can be useful in the clinical settings.
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Long, Lingliang, Yanjun Wu, Lin Wang, Aihua Gong, Rongfeng Hu, and Chi Zhang. "Complete suppression of the fluorophore fluorescence by combined effect of multiple fluorescence quenching groups: A fluorescent sensor for Cu2+ with zero background signals." Analytica Chimica Acta 908 (February 2016): 1–7. http://dx.doi.org/10.1016/j.aca.2015.12.016.
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Taylor, Brad W., Christine F. Keep, Robert O. Hall, Benjamin J. Koch, Lusha M. Tronstad, Alexander S. Flecker, and Amber J. Ulseth. "Improving the fluorometric ammonium method: matrix effects, background fluorescence, and standard additions." Journal of the North American Benthological Society 26, no. 2 (June 2007): 167–77. http://dx.doi.org/10.1899/0887-3593(2007)26[167:itfamm]2.0.co;2.
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Moriyama, Eduardo H., Anthony Kim, Arjen Bogaards, Lothar Lilge, and Brian C. Wilson. "A Ratiometric Fluorescence Imaging System for Surgical Guidance." Advances in Optical Technologies 2008 (October 16, 2008): 1–10. http://dx.doi.org/10.1155/2008/532368.
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A 3-chip CCD imaging system has been developed for quantitative in vivo fluorescence imaging. This incorporates a ratiometric algorithm to correct for the effects of tissue optical absorption and scattering, imaging “geometry” and tissue autofluorescence background. The performance was characterized, and the algorithm was validated in tissue-simulating optical phantoms for quantitative measurement of the fluorescent molecule protoporphyrin IX (PpIX). The technical feasibility to use this system for fluorescence-guided surgical resection of malignant brain tumor tissue was assessed in an animal model in which PpIX was induced exogenously in the tumor cells by systemic administration of aminolevulinic acid (ALA).
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Whalen, Daniel J., and D. Clark Turner. "Effect of X-ray Tube Window Thickness on Detection Limits for Light Elements in XRF Analysis." Advances in X-ray Analysis 38 (1994): 299–305. http://dx.doi.org/10.1154/s0376030800017924.
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Abstract Widespread interest in light element analysis using XRF has stimulated the development of thin x-ray tube windows. Thinner windows enhance the soft x-ray output of the tube, which more efficiently excite the light elements in the sample. A computer program that calculates the effect of window thickness on light element sample fluorescence has been developed. The code uses an NIST algorithm to calculate the x-ray tube spectrum given various tube parameters such as beryllium window thickness, operating voyage, anode composition, and take-off angle. The interaction of the tube radiation with the sample matrix is modelled to provide the primary and secondary fluorescence from the sample. For x-rays in the energy region 30 - 1000 eV the mass attenuation coefficients were interpolated from the photo absorption data compilation of Henke, et al. The code also calculates the x-ray background due to coherent and incoherent scatter from the sample, as well as the contribution of such scatter to the sample fluorescence. Given the sample fluorescence and background the effect of tube window thickness on detection limits for light elements can be predicted.
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Park, Chul-Kyu, and Hoonsung Cho. "Improvement in Tracing Quantum Dot-Conjugated Nanospheres forIn VivoImaging by Eliminating Food Autofluorescence." Journal of Nanomaterials 2015 (2015): 1–6. http://dx.doi.org/10.1155/2015/894353.
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Fluorescence imaging using fluorescent probes has demonstrated long-term stability and brightness suitable forin vivodeep-tissue imaging, but it also allows intense background fluorescence associated with food in the near-infrared (IR) range. We investigated effects of changing rodent diet on food autofluorescence, in the presence of quantum dots-conjugated magnetic nanospheres (QD-MNSs). Replacement of a regular rodent diet with a purified diet has great improvement in removing autofluorescence in the near-infrared range ideal forin vivofluorescence imaging. By feeding a purified diet for eliminating ingredients impairing desirable fluorescence signals in the near-IR range, food autofluorescence was clearly eliminated and fluorescence probes, QD-MNSs, introduced by i.v. injection were effectively traced in a mouse by a distinctive signal-to-noise ratio.
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Atanda, O. O., I. Akpan, E. R. Rati, and M. Ozoje. "Palm Kernel: A Potential Substrate for Rapid Detection of Aflatoxigenic Fungi." Food Science and Technology International 11, no. 1 (February 2005): 67–74. http://dx.doi.org/10.1177/1082013205051293.
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Palm kernel is a cheap natural resource which is abundantly available in the tropics, parts of Asia and South and Central America. A culture medium was developed by incorporating fresh palm kernel extract for the detection of aflatoxigenic fungi. Aflatoxin positive isolates of Aspergilliexhibited a characteristic blue or blue green fluorescence of agar under long wave UV light against a pink background which was confirmed by thin layer chromatography. As compared to conventional desiccated coconut agar, the fluorescent nature of the medium, the intensity and diffusion of the hot water soluble fluorescent compounds of the fungus was unique on this medium. The optimal pH and temperature conditions of aflatoxin production were 7 and 30 ºC respectively. Additives (synthetic and natural) either had no effect or adversely affected the fluorescence of the medium. Aflatoxin detection was possible within 36h in palm kernel broth compared to 40 h in coconut broth. The optimal time of production of fluorescence was 44 h on palm kernel agar compared to 48 h on the conventional medium. Further tests with isolates from different sources showed that yellow pigmentation, fluorescence and aflatoxins were complementary thus obviating the need for UV light. It is thus possible to presumptively identify aflatoxin positive isolates.
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Henry, Charlie, and Paulene O. Roberts. "BACKGROUND FLUORESCENCE VALUES AND MATRIX EFFECTS OBSERVED USING SMART PROTOCOLS IN THE ATLANTIC OCEAN AND GULF OF MEXICO." International Oil Spill Conference Proceedings 2001, no. 2 (March 1, 2001): 1203–7. http://dx.doi.org/10.7901/2169-3358-2001-2-1203.
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ABSTRACT For a 6-month period between May and September 1999, the staff and crew of the National Oceanic and Atmospheric Administration (NOAA) research ship R/V Ferrel augmented their activities to support a baseline fluorometry study for NOAA's Office of Response and Restoration (OR&R). During the study period, scientific data were collected at more than 50 stations in the Atlantic Ocean and Gulf of Mexico. The study was designed, in part, to assess the Special Monitoring of Applied Response Technologies (SMART) program. SMART is a joint U.S. Coast Guard (USCG), U.S. Environmental Protection Agency (EPA), Centers for Disease Control and Prevention (CDC), and NOAA monitoring program that provides near real-time feedback to the Unified Command during dispersant applications to mitigate marine oil spills. Using fluorometers to accurately measure dispersed oil concentrations is not a trivial task. Detector response values vary because of oil composition and oil weathering; dispersed oil is not in true solution; and natural waters contribute matrix effects and background fluorescence. It was the latter two that were investigated. Because seawater is a complex mixture of dissolved chemicals, particulates, and living plants and animals, archiving samples for future analysis relative to background fluorescence is problematic. Each contributes to background fluorescence and matrix effects when dispersed oil is present in the sample. Since water samples change with storage, only near real-time analyses are valid; therefore, using an at-sea laboratory like the NOAA's Ferrel was essential to the study design. Results suggest that the adverse flouorometry effects observed for open-ocean environments were within typical quality-objective goals. Therefore, the range of seawater composition changes observed in this investigation had very little effect on the ability to detect dispersed oil and meet the SMART mission objectives.
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Liang, Ying, Yingming Pan, Qing Guo, Hongzhi Hu, Chancui Wu, and Qian Zhang. "A Novel Analytical Method for Trace Ammonium in Freshwater and Seawater Using 4-Methoxyphthalaldehyde as Fluorescent Reagent." Journal of Analytical Methods in Chemistry 2015 (2015): 1–7. http://dx.doi.org/10.1155/2015/387207.
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A novel fluorescent reagent for determination of ammonium, 4-methoxyphthalaldehyde (MOPA), was successfully synthesized in this study. Under alkaline conditions, MOPA could reacted with ammonium rapidly at room temperature, producing fluorescent substance which had maximum excitation at 370 nm and emission wavelength at 454 nm. Based on this, a novel fluorescence analysis method was established for the determination of trace ammonium in natural water. Experimental parameters including reagent concentration, pH, reaction equilibrium time, and metal ions masking agent were optimized. The results showed that the optimized MOPA concentration was 0.12 g/L, pH was in the range of 11.2–12.0, and sulfite concentration was 0.051 g/L, respectively. Metal ions masking agent had no obvious effect on the fluorescence signal. With the reaction time of 15 minutes, linear range of this method was between 0.025 and 0.300 μmol/L, and the method detecting limit was 0.0058 μmol/L. The matrix recovery of the proposed method was in the range of 93.6–108.1%. Compared with the OPA method, this method was much more sensitive and rapid without the interference of background peak and would be more suitable for developing a portable fluorescence detection system.
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Leisey, J. R., D. A. Scott, L. W. Grotyohann, and R. C. Scaduto. "Quantitation of myoglobin saturation in the perfused heart using myoglobin as an optical inner filter." American Journal of Physiology-Heart and Circulatory Physiology 267, no. 2 (August 1, 1994): H645—H653. http://dx.doi.org/10.1152/ajpheart.1994.267.2.h645.
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Quantitation of metabolic parameters using the technique of cardiac surface fluorescence is complicated by motion and changes in tissue absorption. Because ratio fluorescence methodology can be applied to eliminate motion-induced errors, in the current study, we used a ratio fluorescence technique to evaluate myoglobin saturation in the perfused rat heart, since myoglobin is the major oxygen-dependent light absorbing species in this tissue. Changes in myoglobin saturation can affect surface fluorescence measurements as a result of the inner filter effect. Optical scans of heart extracts indicated the major absorption peak is due to myoglobin and its peak wavelength shifts from 415 to 430 nm upon deoxygenation. To monitor this shift in hearts, the isolated perfused heart was loaded covalently with the fluorescent dye 7-diethylaminocoumarin-3-carboxylic acid by brief perfusion with the succinimidyl ester. This dye has an excitation maximum in the region of maximal absorption by myoglobin and allows for monitoring myoglobin oxygenation using the inner filter effect. The dye localized to endothelial cells and increased the surface fluorescence in this wavelength region approximately 50-fold above background levels without affecting cardiac function. An equation was derived to estimate the fraction of myoglobin in the oxygenated state from changes in the fluorescence 415/430 excitation ratio. From this fraction, the average PO2 in the environment of myoglobin was estimated under several perfusion conditions. We report that retrograde perfusion in the Langendorff mode at either 60 or 120 mmHg pressure resulted in full oxygenation of myoglobin with use of cell-free perfusate equilibrated with 95% O2.(ABSTRACT TRUNCATED AT 250 WORDS)
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Zhang, Chong, Daqing Jiang, Bo Huang, Cong Wang, Lin Zhao, Xianxin Xie, Zhaohe Zhang, Kun Wang, Jie Tian, and Yahong Luo. "Methylene Blue–Based Near-Infrared Fluorescence Imaging for Breast Cancer Visualization in Resected Human Tissues." Technology in Cancer Research & Treatment 18 (January 1, 2019): 153303381989433. http://dx.doi.org/10.1177/1533033819894331.
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Breast-conserving surgery is facing the challenge of objective tumor margin identification intraoperatively. Near-infrared fluorescence imaging would be an ideal approach to visualize tumor margins during surgeries. In this preliminary study, the feasibility of methylene blue–based near-infrared fluorescence imaging technique for breast cancer detection was assessed in resected human breast specimens after breast cancer surgeries. Thirty patients with breast cancer scheduled for surgical treatment were enrolled, including 10 patients with preoperative chemotherapy and 20 patients without. Each of them received an injection of 1 mg/kg methylene blue intravenously 3 hours before the surgery. Then, a home-developed methylene blue–specific near-infrared fluorescence imaging system was employed to image the resected breast tissues and identify the tumor by the fluorescence contrast. Specimens were taken for pathological examinations as the reference. There were no severe adverse events attributable to methylene blue. Of 20 patients, who did not receive preoperative chemotherapy, 16 exhibited fluorescent contrast on their resected tissues (signal-to-background ratio: 1.94 ± 0.71). In contrast, tumors were identified in 3 of 10 specimens from patients who underwent preoperative chemotherapy (signal-to-background ratio: 1.63 ± 0.38). A total of 35 tissues were sampled from 30 specimens. Besides 30 tumor samples, 5 more suspicious samples with fluorescence signal were confirmed to be benign hemorrhagic tissues. Therefore, a sensitivity of 0.63 and a positive predictive value of 0.79 were achieved by the methylene blue fluorescence imaging strategy. Here, we demonstrate the feasibility of using methylene blue fluorescence imaging to identify breast cancer. Preoperative chemotherapy had an impact on imaging effect, which may reduce the detection rate. After all, methylene blue fluorescence imaging has great potential to be used into breast-conserving surgery for tumor-positive margins detection, but further clinical trial study is needed ( http://www.chictr.org.cn/ Clinical Trial Registry ID: ChiCTR1800015400, Near-infrared fluorescence imaging applied in breast cancer identification with methylene blue).
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Bissinger, Rosi, Abdulla Al Mamun Bhuyan, Elena Signoretto, and Florian Lang. "Stimulating Effect of Elvitegravir on Suicidal Erythrocyte Death." Cellular Physiology and Biochemistry 38, no. 3 (2016): 1111–20. http://dx.doi.org/10.1159/000443061.
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Background/Aims: The antiviral drug Elvitegravir is used for the treatment of Human Immunodeficiency Virus (HIV) infections. The present study explored whether the drug is able to trigger eryptosis, the suicidal death of erythrocytes. Eryptosis is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, activated p38 kinase and activated caspases. The present study explored, whether Elvitegravir induces eryptosis and, if so, to shed light on the mechanisms involved. Methods: Phosphatidylserine abundance at the erythrocyte surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, abundance of reactive oxygen species (ROS) from DCFDA dependent fluorescence, and ceramide abundance at the erythrocyte surface utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to Elvitegravir (≥ 1.5 µg/ml) significantly increased the percentage of annexin-V-binding cells, and significantly decreased forward scatter. Elvitegravir (2.5 µg/ml) significantly increased Fluo3-fluorescence, but did not significantly modify DCFDA fluorescence or ceramide abundance. The effect of Elvitegravir on annexin-V-binding was significantly blunted by removal of extracellular Ca2+, but not in the presence of p38 kinase inhibitor SB203580 (2 µM) or in the presence of pancaspase inhibitor zVAD (10 µM). Conclusions: Elvitegravir triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to entry of extracellular Ca2+.
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Diaspro, Alberto, Giuseppe Chirico, and Maddalena Collini. "Two-photon fluorescence excitation and related techniques in biological microscopy." Quarterly Reviews of Biophysics 38, no. 2 (May 2005): 97–166. http://dx.doi.org/10.1017/s0033583505004129.
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1. Introduction 982. Historical background of two-photon effects 992.1 2PE 1002.2 Harmonic generation 1002.3 Fluorescence correlation spectroscopy 1003. Basic principles of two-photon excitation of fluorescent molecules and implications for microscopy and spectroscopy 1013.1 General considerations 1013.2 Fluorescence intensity under the 2PE condition 1033.3 Optical consequences of 2PE 1043.4 Saturation effects in 2PE 1083.5 Fluorescence correlation spectroscopy 1093.5.1 Autocorrelation analysis 1103.5.2 Photon-counting histogram analysis 1124. Two-photon-excited probes 1155. Design considerations for a 2PE fluorescence microscope 1195.1 General aspects 1195.2 Descanned and non-descanned 2PE imaging 1215.3 Lens objectives and pulse broadening 1225.4 Laser sources 1255.5 Example of a practical realization 1276. Applications 1346.1 Biological applications of 2PE 1346.1.1 Brain images 1346.1.2 Applications on the kidney 1396.1.3 Mammalian embryos 1396.1.4 Applications to immuno-response 1416.1.5 Myocytes 1416.1.6 Retina 1426.1.7 DNA imaging 1436.1.8 FISH applications 1446.2 2PE imaging of single molecules 1446.3 FCS applications 1486.4 Signals from nonlinear interactions 1517. Conclusions 1538. Acknowledgements 1549. References 155This review is concerned with two-photon excited fluorescence microscopy (2PE) and related techniques, which are probably the most important advance in optical microscopy of biological specimens since the introduction of confocal imaging. The advent of 2PE on the scene allowed the design and performance of many unimaginable biological studies from the single cell to the tissue level, and even to whole animals, at a resolution ranging from the classical hundreds of nanometres to the single molecule size. Moreover, 2PE enabled long-term imaging of in vivo biological specimens, image generation from deeper tissue depth, and higher signal-to-noise images compared to wide-field and confocal schemes. However, due to the fact that up to this time 2PE can only be considered to be in its infancy, the advantages over other techniques are still being evaluated. Here, after a brief historical introduction, we focus on the basic principles of 2PE including fluorescence correlation spectroscopy. The major advantages and drawbacks of 2PE-based experimental approaches are discussed and compared to the conventional single-photon excitation cases. In particular we deal with the fluorescence brightness of most used dyes and proteins under 2PE conditions, on the optical consequences of 2PE, and the saturation effects in 2PE that mostly limit the fluorescence output. A complete section is devoted to the discussion of 2PE of fluorescent probes. We then offer a description of the central experimental issues, namely: choice of microscope objectives, two-photon excitable dyes and fluorescent proteins, choice of laser sources, and effect of the optics on 2PE sensitivity. An inevitably partial, but vast, overview of the applications and a large and up-to-date bibliography terminate the review. As a conclusive comment, we believe that 2PE and related techniques can be considered as a mainstay of the modern biophysical research milieu and a bright perspective in optical microscopy.
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Kozubenko, E. A., P. A. Zykin, E. I. Krasnoshchekova, L. A. Tkachenko, K. N. Fedoseeva, and A. D. Kharazova. "Method of reduction background fluorescence in human fetal brain tissue and quantitative estimate of the effect of photobleaching." Bulletin of Experimental Biology and Medicine 171, no. 1 (2021): 122–28. http://dx.doi.org/10.47056/0365-9615-2021-171-1-122-128.
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Jenvey, Caitlin J., and Judith R. Stabel. "Autofluorescence and Nonspecific Immunofluorescent Labeling in Frozen Bovine Intestinal Tissue Sections: Solutions for Multicolor Immunofluorescence Experiments." Journal of Histochemistry & Cytochemistry 65, no. 9 (August 1, 2017): 531–41. http://dx.doi.org/10.1369/0022155417724425.
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Autofluorescent compounds present in intestinal tissue often hinder the ability to utilize multiple, spectrally different, fluorophores. In addition, fixatives and blocking solutions may contribute to background autofluorescence or nonspecific immunofluorescent labeling. During immunofluorescence protocol development, autofluorescent pigments were observed in frozen bovine mid-ileal intestinal tissue sections. Coagulant fixatives, normal serum blocking, histochemical stains Sudan Black B (SBB) and 3,3′-diaminobenzidine (DAB), and spectral separation using imaging software were compared for their ability to reduce autofluorescence, as well as their effect on immunofluorescent labeling. Fluorescent pigments of frozen bovine mid-ileal intestinal tissue sections, most likely caused by eosinophils and lipofuscin, were masked successfully with a combination of DAB and SBB. Little to no statistical differences were observed for all other methods investigated; however, tissue fixed with 1:1 acetone methanol and 10% horse serum diluted in 0.05 M Tris buffer demonstrated lower mean fluorescence intensities. Spectral separation of specific immunofluorescent labeling from background autofluorescence is a simple method for removing unwanted fluorescence; however, successful separation is dependent on tissue and labeling quality.
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Liu, Jin-Xia, Mei-Xia Wu, and Shou-Nian Ding. "Aggregation-Induced Emission Enhancement of CdSe QDs by Protamine and its Application to Sensitively and Selectively Detect Heparin." Current Analytical Chemistry 15, no. 5 (July 12, 2019): 599–604. http://dx.doi.org/10.2174/1573411014666180330160743.
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Background: Heparin, it is commercially used as an anticoagulant in surgical procedures for the prevention of blood clotting. However, overdose and prolonged use of heparin often induce potentially fatal bleeding complication. So, it is of crucial importance to monitor closely heparin levels for the sake of health. In this work, a sensitive fluorescence sensing platform to detect heparin was set up based on MPA-CdSe QDs (quantum dots) and protamine enhanced fluorescent system. Methods: The image of CdSe QDs was taken on a JEM-2100 transmission electron microscope (JEOL Ltd.). The fluorescence spectrum was recorded on a FluoroMax-4 fluorescence spectrophotometer (Horiba, USA). UV–vis absorption spectrum was recorded using a Shimadzu UV-2450 Spectrophotometer (Tokyo, Japan). A vortex mixer IKA MS3 digital was selected to mix the solution. Results: Under optimized conditions, the linear response to detect heparin ranges from 0.06 to 14 µg mL-1 with a detection limit of 8 ng mL-1. The approach showed a highly selective response to heparin in the presence of 16 interfered substances. Conclusion: A simple method for the detection of heparin was developed based on MPA-CdSe QDs and protamine enhanced fluorescent system. The electrostatic effect between MPA-CdSe QDs and protamine resulted in strong fluorescence enhancement from the MPA-CdSe QDs. Moreover, the addition of heparin could cause a significant fluorescence decrease due to the strong affinity of protamine and heparin. Under optimal conditions, this method displayed a low detection limit and good selectivity over other substances.
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Soleimaninejad, H., F. Matroodi, and S. H. Tavassoli. "Raman Spectroscopy of Iranian Region Calcite Using Pulsed Laser: An Approach of Fluorescence Suppression by Time-Gating Method." Journal of Spectroscopy 2013 (2013): 1–6. http://dx.doi.org/10.1155/2013/254964.
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The effect of time-gating method in Raman spectroscopy for fluorescence suppression of Iranian region calcite is investigated. Experiments are done using an Nd:YAG laser with a pulse durations of 10 ns at wavelength 532 nm. Seven samples from different places are examined. In order to obtain the optimum gate width for fluorescence suppression, a series of experiments is carried out at different gate widths. Raman-to-fluorescence (R/F) and fluorescence-to-laser peak (F/L) ratios are compared at gated and nongated experiments. Applying the optimum gate width leads to an effective reduction of fluorescence background and improvement in both ratios of R/F and F/L. Raman signals of some samples in nongated experiments are completely hidden by fluorescence while emerged in gated experiments.
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Mischitelli, Morena, Mohamed Jemaà, Mustafa Almasry, Caterina Faggio, and Florian Lang. "Stimulation of Erythrocyte Cell Membrane Scrambling by Quinine." Cellular Physiology and Biochemistry 40, no. 3-4 (2016): 657–67. http://dx.doi.org/10.1159/000452578.
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Background/Aims: The analkaloid drug quinine is utilized mainly for the chemoprophylaxis of malaria. The multiple side effects of quinine include hemolytic anemia and hemolytic uremic syndrome, disorders involving suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling contributing to stimulation of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide and D4476 sensitive casein kinase. The present study explored the putative effect of quinine on eryptosis and elucidated cellular mechanisms involved. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCF dependent fluorescence, and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to quinine (≥ 50 µM) significantly increased the percentage of annexin-V-binding cells without significantly affecting forward scatter. Quinine significantly increased Fluo3-fluorescence, DCF fluorescence and ceramide abundance. The effect of quinine on annexin-V-binding was significantly blunted by removal of extracellular Ca2+ and by addition of D4476 (10 µM). Conclusions: Quinine triggers phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca2+ entry, oxidative stress, ceramide and D4476 sensitive casein kinase.
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Zhang, Mingxi, Jingying Yue, Ran Cui, Zhuoran Ma, Hao Wan, Feifei Wang, Shoujun Zhu, et al. "Bright quantum dots emitting at ∼1,600 nm in the NIR-IIb window for deep tissue fluorescence imaging." Proceedings of the National Academy of Sciences 115, no. 26 (June 11, 2018): 6590–95. http://dx.doi.org/10.1073/pnas.1806153115.
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With suppressed photon scattering and diminished autofluorescence, in vivo fluorescence imaging in the 1,500- to 1,700-nm range of the near-IR (NIR) spectrum (NIR-IIb window) can afford high clarity and deep tissue penetration. However, there has been a lack of NIR-IIb fluorescent probes with sufficient brightness and aqueous stability. Here, we present a bright fluorescent probe emitting at ∼1,600 nm based on core/shell lead sulfide/cadmium sulfide (CdS) quantum dots (CSQDs) synthesized in organic phase. The CdS shell plays a critical role of protecting the lead sulfide (PbS) core from oxidation and retaining its bright fluorescence through the process of amphiphilic polymer coating and transferring to water needed for imparting aqueous stability and compatibility. The resulting CSQDs with a branched PEG outer layer exhibited a long blood circulation half-life of 7 hours and enabled through-skin, real-time imaging of blood flows in mouse vasculatures at an unprecedented 60 frames per second (fps) speed by detecting ∼1,600-nm fluorescence under 808-nm excitation. It also allowed through-skin in vivo confocal 3D imaging of tumor vasculatures in mice with an imaging depth of ∼1.2 mm. The PEG-CSQDs accumulated in tumor effectively through the enhanced permeation and retention effect, affording a high tumor-to-normal tissue ratio up to ∼32 owing to the bright ∼1,600-nm emission and nearly zero autofluorescence background resulting from a large ∼800-nm Stoke’s shift. The aqueous-compatible CSQDs are excreted through the biliary pathway without causing obvious toxicity effects, suggesting a useful class of ∼1,600-nm emitting probes for biomedical research.
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Sripathi, Punchithaya K., and K. M. Balakrishna. "K-Shell X-Ray Fluorescence Studies of Some Elements in the Atomic Number Range 26 = Z = 70." International Journal of Chemoinformatics and Chemical Engineering 3, no. 2 (July 2013): 27–44. http://dx.doi.org/10.4018/ijcce.2013070102.
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K-shell fluorescence yields of low, medium Z and rare earth elements were determined using Si(PIN) detector and HPGe detector employing reflection geometry set up. Target atoms were excited using 59.5 keV gamma rays emerging from Am-241 source of strength 300 mCi. Background radiation and multiple scattering effects were minimized by properly shielding the detector. The elemental foils of uniform thickness and 99.9% purity were used in the present investigation. The fluorescent spectra were recorded in an 8K and 16K multi channel analyzer. The data were carefully analyzed and total K-shell fluorescence yields were calculated. The resulting yield values are compared with the available experimental and theoretical values.
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Li, Xiaodong. "Preparation of Graphene Oxide and Its Application as Substrates for SERS." Journal of Chemistry 2018 (October 17, 2018): 1–5. http://dx.doi.org/10.1155/2018/8050524.
Full textAbstract:
Graphene oxide (GO) was synthesized by a modified Hummer’s method and was then reduced by the hydrothermal process. Both GO and reduced GO (rGO) were employed as surface-enhanced Raman scattering (SERS) substrates to detect rhodamine 6G (R6G). After adsorbed on the surface of GO, the fluorescence background of R6G was highly quenched, and its Raman scatterings were enhanced. When adsorbed on the surface of rGO, the fluorescence background was further quenched at the price of lower SERS intensity. The results displayed that oxygen groups on the surface of GO had positive effect on the SERS effect of GO. The amount of oxygen groups on the surface of GO should be one key parameter to adjust the SERS activity of GO.
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Signoretto, Elena, Michela Castagna, Abdulla Al Mamun Bhuyan, and Florian Lang. "Stimulating Effect of Terfenadine on Erythrocyte Cell Membrane Scrambling." Cellular Physiology and Biochemistry 38, no. 4 (2016): 1425–34. http://dx.doi.org/10.1159/000443085.
Full textAbstract:
Background/Aims: The antihistaminic drug Terfenadine may trigger apoptosis of tumor cells, an effect unrelated to its effect on histamine receptors. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal death of erythrocytes characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling triggering eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, and ceramide. The present study explored, whether Terfenadine is capable to trigger eryptosis. Methods: Flow cytometry was employed to estimate phosphatidylserine abundance at the erythrocyte surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, abundance of reactive oxygen species (ROS) from 2′,7′-dichlorodihydrofluorescein (DCF) diacetate dependent fluorescence, and ceramide abundance at the human erythrocyte surface utilizing specific antibodies. Hemolysis was quantified from haemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to Terfenadine (≥ 5 µM) significantly increased the percentage of annexin-V-binding cells and triggered hemolysis without significantly modifying the average forward scatter. Terfenadine (7.5 µM) significantly increased Fluo3-fluorescence, but did not significantly modify DCF fluorescence or ceramide abundance. The effect of Terfenadine on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca2+. Exposure of human erythrocytes to Ca2+ ionophore ionomycin (1 µM, 15 min) triggered annexin-V-binding, an effect augmented by Terfenadine pretreatment (10 µM, 48 hours). Conclusions: Terfenadine triggers phospholipid scrambling of the human erythrocyte cell membrane, an effect in part due to entry of extracellular Ca2+ and in part due to sensitizing human erythrocyte cell membrane scrambling to Ca2+.
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Mii, Sumiyuki, Yasunori Tome, Yukihiko Hiroshima, Fuminari Uehara, Shinji Miwa, Toshiyoshi Fujiwara, Robert M. Hoffman, and Shuya Yano. "Effect of dormant cancer cells on angiogenesis after resisting chemotherapy." Journal of Clinical Oncology 31, no. 15_suppl (May 20, 2013): e13577-e13577. http://dx.doi.org/10.1200/jco.2013.31.15_suppl.e13577.
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e13577 Background: Dormant cancer cells are a significant problem in clinical cancer as they are usually chemoresistant and can give rise to recurrence years after treatment. Methods: Dormant cancer cells were identified with a fluorescence ubiquitination cell cycle indicator (FUCCI) and confocal microscopy. FUCCI-expressing xenografts were established from MKN45 gastric cancer cells. Results: Within 7 days after implantation in nude mice, FUCCI-expressing tumors contained a mixture of cancer cells in G0/G1 and S/G2/M phases. Cancer cells in G0/G1 phase survived and become dormant after treatment with cytotoxic agents. As the tumors grew, proliferating cells were only found at the surface. Chemotherapy killed only the proliferating cancer cells at the surface and had little effect on the majority of dormant cancer cells in the tumor center. Furthermore, we investigated tumor chemoresistance using nestin-driven green fluorescent protein (GFP) transgenic mice that have GFP-expressing nascent tumor vessels. Chemotherapy-treated tumors had much more and deeper tumor vessels than control tumors. Conclusions: These results suggest that dormant cancer cells may protect themselves by inducing angiogenesis.
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Rosenberg, Adrian, Daiki Fujimura, Ryuhei Okada, Aki Furusawa, Fuyuki Inagaki, Hiroaki Wakiyama, Takuya Kato, Peter L. Choyke, and Hisataka Kobayashi. "Real-Time Fluorescence Imaging Using Indocyanine Green to Assess Therapeutic Effects of Near-Infrared Photoimmunotherapy in Tumor Model Mice." Molecular Imaging 19 (January 1, 2020): 153601212093496. http://dx.doi.org/10.1177/1536012120934965.
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Background: Near-infrared photoimmunotherapy (NIR-PIT) is a cancer therapy that causes an increase in tumor perfusion, a phenomenon termed the super-enhanced permeability and retention effect. Currently, in vivo treatment efficacy of NIR-PIT is observable days after treatment, but monitoring would be improved by more acute detection of intratumor change. Fluorescence imaging may detect increased tumor perfusion immediately after treatment. Methods: In the first experiment, athymic nude mouse models bearing unilateral subcutaneous flank tumors were treated with either NIR-PIT or laser therapy only. In the second experiment, mice bearing bilateral flank tumors were treated with NIR-PIT only on the left-sided tumor. In both groups, immediately after treatment, indocyanine green was injected at different doses intravenously, and mice were monitored with the Shimadzu LIGHTVISION fluorescence imaging system for 1 hour. Results: Tumor-to-background ratio of fluorescence intensity increased over the 60 minutes of monitoring in treated mice but did not vary significantly in control mice. Tumor-to-background ratio was highest in the 1 mg kg−1 and 0.3 mg kg−1 doses. In mice with bilateral tumors, tumor-to-untreated tumor ratio increased similarly. Conclusions: Acute changes in tumor perfusion after NIR-PIT can be detected by real-time fluorescence imaging.
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Kohro, Shinji, Quinn H. Hogan, Yuri Nakae, Michiaki Yamakage, and Zeljko J. Bosnjak. "Anesthetic Effects on Mitochondrial ATP-sensitive K Channel." Anesthesiology 95, no. 6 (December 1, 2001): 1435–40. http://dx.doi.org/10.1097/00000542-200112000-00024.
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Background Volatile anesthetics show an ischemic preconditioning-like cardioprotective effect, whereas intravenous anesthetics have cardioprotective effects for ischemic-reperfusion injury. Although recent evidence suggests that mitochondrial adenosine triphosphate-regulated potassium (mitoK(ATP)) channels are important in cardiac preconditioning, the effect of anesthetics on mitoK(ATP) is unexplored. Therefore, the authors tested the hypothesis that anesthetics act on the mitoK(ATP) channel and mitochondrial flavoprotein oxidation. Methods Myocardial cells were isolated from adult guinea pigs. Endogenous mitochondrial flavoprotein fluorescence, an indicator of mitochondrial flavoprotein oxidation, was monitored with fluorescence microscopy while myocytes were exposed individually for 15 min to isoflurane, sevoflurane, propofol, and pentobarbital. The authors further investigated the effect of 5-hydroxydeanoate, a specific mitoK(ATP) channel antagonist, on isoflurane- and sevoflurane-induced flavoprotein oxidation. Additionally, the effects of propofol and pentobarbital on isoflurane-induced flavoprotein oxidation were measured. Results Isoflurane and sevoflurane induced dose-dependent increases in flavoprotein oxidation (isoflurane: R2 = 0.71, n = 50; sevoflurane: R2 = 0.86, n = 20). The fluorescence increase produced by both isoflurane and sevoflurane was eliminated by 5-hydroxydeanoate. Although propofol and pentobarbital showed no significant effects on flavoprotein oxidation, they both dose-dependently inhibited isoflurane-induced flavoprotein oxidation. Conclusions Inhalational anesthetics induce flavoprotein oxidation through opening of the mitoK(ATP) channel. This may be an important mechanism contributing to anesthetic-induced preconditioning. Cardioprotective effects of intravenous anesthetics may not be dependent on flavoprotein oxidation, but the administration of propofol or pentobarbital may potentially inhibit the cardioprotective effect of inhalational anesthetics.
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Signoretto, Elena, Stefan A. Laufer, and Florian Lang. "Stimulating Effect of Sclareol on Suicidal Death of Human Erythrocytes." Cellular Physiology and Biochemistry 39, no. 2 (2016): 554–64. http://dx.doi.org/10.1159/000445647.
Full textAbstract:
Background/Aims: The diterpene alcohol Sclareol has been proposed for the treatment of malignancy. In analogy to apoptosis of nucleated cells, erythrocytes may enter eryptosis, a suicidal cell death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Cellular mechanisms involved in the triggering of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, p38 kinase and casein kinase 1α. The present study explored, whether Sclareol induces eryptosis and, if so, shed light on the mechanisms involved. Methods: Phosphatidylserine abundance at the erythrocyte surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, abundance of reactive oxygen species (ROS) from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA)-dependent fluorescence, and ceramide abundance at the erythrocyte surface utilizing specific antibodies. Hemolysis was estimated from haemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to Sclareol (≥ 50 µM) significantly increased the percentage of annexin-V-binding cells without significantly modifying the average forward scatter, DCF-fluorescence or ceramide abundance. Sclareol (≥ 50 µM) further triggered hemolysis. Sclareol (100 µM) significantly increased Fluo3-fluorescence, but the effect of Sclareol on annexin-V-binding was not significantly blunted by removal of extracellular Ca2+. Instead, the effect of Sclareol on annexin-V-binding was significantly blunted in the presence of p38 kinase inhibitor skepinone (2 µM) and in the presence of casein kinase 1α inhibitor D4476 (10 µM). Conclusions: Sclareol triggers phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to activation of p38 kinase and casein kinase 1α.
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Fehér, E., B. Major, K. Bélafi-Bakó, and L. Gubicza. "On the background of enhanced stability and reusability of enzymes in ionic liquids." Biochemical Society Transactions 35, no. 6 (November 23, 2007): 1624–27. http://dx.doi.org/10.1042/bst0351624.
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The ability of ILs (ionic liquids) to provide an environment of increased stability and, in this way, improve the recyclability of enzymes has been studied. The description of this phenomenon is not easy; there are several approaches for explanation. In this mini-review, the results from different research groups are summarized, with the aim of explaining the strong stability effect of ILs on several enzymes. Spectroscopic methods (e.g. fluorescence and CD, IR spectroscopy, mass spectroscopy and NMR) and investigations of polarity and kosmotropicity of ions are promising methods. Since higher stability means that we may be able to reuse enzymes more times, the recyclability of enzymes was also in the focus. From this point of view, the advantages and disadvantages of applying monophasic or biphasic systems are discussed too, presenting the coupled techniques as well.
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Mines, C. H., A. Ghadouani, and G. N. Ivey. "Dying to find the source – the use of rhodamine WT as a proxy for soluble point source pollutants in closed pipe surface drainage networks." Hydrology and Earth System Sciences 13, no. 11 (November 12, 2009): 2169–78. http://dx.doi.org/10.5194/hess-13-2169-2009.
Full textAbstract:
Abstract. Rhodamine WT (RWT), a xanthene dye, may serve as a proxy for soluble pollutants within quantitative tracing studies investigating point source contaminant transport. This study quantified the effects of altering the concentration, pH, temperature and salinity of a RWT solution on the detected fluorescence of RWT within the laboratory prior to a field release of RWT within a closed pipe urban drainage network. All RWT solutions exhibited stability and <10% variation from the expected concentration over a thirteen hour laboratory study period; pH related quenching of RWT fluorescence of up to 14.9% was observed for solutions with pH<3.9; and increasing salinity of RWT solution was found to have a negligible quenching effect. In direct contrast to previous studies RWT fluorescence was found to directly correlate with temperature of solution, and a temperature correction factor was determined and tested. The field release study succeeded in detecting RWT at concentrations two orders of magnitude greater than background fluorescence. Based on longitudinal dispersion theory, observed RWT peak concentrations were within 10% of predicted peaks.
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Zhang, Chunling, Liying Zhang, Ru Yang, Kun Liang, and Dejun Han. "Time-Correlated Raman and Fluorescence Spectroscopy Based on a Silicon Photomultiplier and Time-Correlated Single Photon Counting Technique." Applied Spectroscopy 67, no. 2 (February 2013): 136–40. http://dx.doi.org/10.1366/12-06736.
Full textAbstract:
We report a time-correlated Raman spectroscopy technique based on a silicon photomultiplier (SiPM) and a time-correlated single photon counting (TCSPC) technique to exploit the natural temporal separation between Raman and fluorescence phenomena to alleviate the high fluorescence background with conventional Raman detection. The TCSPC technique employed can greatly reduce the effect of high dark count rate (DCR) and crosstalk of SiPM that seriously hinder its application in low light level detection. The operating principle and performance of the 400 ps time resolution system are discussed along with the improvement of the peak-to-background ratio (PBR) for bulk trinitrotoluene (TNT) Raman spectrum relative to a commercial Raman spectrometer with charge coupled device (CCD). The fluorescence lifetime for solid TNT and Surface Enhanced Raman Scattering (SERS) spectrum for 10−6 mol/L trace TNT have also been obtained by this system, showing excellent versatility and convenience in spectroscopy measurement.
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Signoretto, Elena, Rosi Bissinger, Michela Castagna, and Florian Lang. "Stimulation of Eryptosis by Combretastatin A4 Phosphate Disodium (CA4P)." Cellular Physiology and Biochemistry 38, no. 3 (2016): 969–81. http://dx.doi.org/10.1159/000443049.
Full textAbstract:
Background/Aims: Combretastatin A4 phosphate disodium (CA4P) is utilized for the treatment of malignancy. The substance has previously been shown to trigger suicidal cell death or apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), ceramide, oxidative stress and ATP depletion. The present study explored, whether CA4P induces eryptosis and, if so, to gain insight into mechanisms involved. Methods: Flow cytometry has been employed to estimate phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) abundance from DCF fluorescence, glutathione (GSH) abundance from CMF fluorescence and ceramide abundance from fluorescent antibodies. In addition cytosolic ATP levels were quantified utilizing a luciferin-luciferase-based assay and hemolysis was estimated from hemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to CA4P (≥ 50 µM) significantly increased the percentage of annexin-V-binding cells and significantly decreased forward scatter. CA4P did not appreciably increase hemolysis. Hundred µM CA4P significantly increased Fluo3-fluorescence. The effect of CA4P (100 µM) on annexin-V-binding was significantly blunted, but not abolished, by removal of extracellular Ca2+. CA4P (≥ 50 µM) significantly decreased GSH abundance and ATP levels but did not significantly increase ROS or ceramide. Conclusions: CA4P triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to entry of extracellular Ca2+ and energy depletion.
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Al Mamun Bhuyan, Abdulla, and Florian Lang. "Stimulation of Eryptosis by Afatinib." Cellular Physiology and Biochemistry 47, no. 3 (2018): 1259–73. http://dx.doi.org/10.1159/000490221.
Full textAbstract:
Background/Aims: The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor afatinib is primarily utilized for the treatment of non-small cell lung carcinoma. The drug is at least partially effective by triggering suicidal tumor cell death. Side effects of afatinib treatment include anemia. At least in theory, afatinib induced anemia could be secondary to stimulation of suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling potentially stimulating eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), induction of oxidative stress, and increase of ceramide abundance. The present study explored, whether afatinib induces eryptosis and, if so, whether its effect involves Ca2+ entry, oxidative stress, and/or ceramide. Methods: Flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) abundance from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to afatinib (≥ 4 µg/ml) significantly increased the percentage of annexin-V-binding cells and significantly decreased forward scatter. Afatinib significantly increased Fluo3-fluorescence, DCFDA fluorescence and ceramide abundance. The effect of afatinib on annexin-V-binding and forward scatter was significantly blunted by removal of extracellular Ca2+. Conclusions: Afatinib triggers phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca2+ entry, oxidative stress, and ceramide.
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Briglia, Marilena, Antonella Fazio, Caterina Faggio, Stefan Laufer, Kousi Alzoubi, and Florian Lang. "Triggering of Suicidal Erythrocyte Death by Ruxolitinib." Cellular Physiology and Biochemistry 37, no. 2 (2015): 768–78. http://dx.doi.org/10.1159/000430394.
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Background/Aims: The JAK1/JAK2 tyrosine kinase inhibitor ruxolitinib is widely used for the treatment of myeloproliferative neoplasm-associated myelofibrosis and other malignancies. Most important side effects include anemia. A common cause of anemia is accelerated suicidal death of erythrocytes or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Mechanisms contributing to the triggering of eryptosis include oxidative stress, Ca2+ entry with increase of cytosolic Ca2+ activity ([Ca2+]i), and activation of distinct kinases, such as p38 mitogen activated protein (MAP) kinase. The present study explored whether and how ruxolitinib induces eryptosis. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, hemolysis from hemoglobin release, [Ca2+]i from Fluo3-fluorescence, and ROS formation from DCFDA dependent fluorescence. Results: A 48 hours exposure of human erythrocytes to ruxolitinib (25 µM) significantly increased the percentage of annexin-V-binding cells and significantly decreased forward scatter. Ruxolitinib did not significantly modify Fluo3-fluorescence and DCFDA fluorescence and the effect of ruxolitinib on annexin-V-binding was not significantly modified by removal of extracellular Ca2+. The effect of ruxolitinib on annexin-V-binding was, however, significantly blunted by the p38 MAP kinase inhibitor SB203580 and virtually abolished by the p38 MAP kinase inhibitor skepinone. Conclusion: Ruxolitinib triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part requiring p38 MAP kinase activity.
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Al Mamun Bhuyan, Abdulla, Teresa Wagner, Hang Cao, and Florian Lang. "Triggering of Suicidal Erythrocyte Death by Gefitinib." Cellular Physiology and Biochemistry 41, no. 4 (2017): 1697–708. http://dx.doi.org/10.1159/000471823.
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Background/Aims: The epidermal growth factor receptor-tyrosine kinase inhibitor gefitinib is effective against several malignancies and is mainly utilized in the treatment of epidermal growth factor receptor mutation positive non-small cell lung cancer. The anti-cancer effect of the drug involves stimulation of apoptosis. Side effects of gefitinib include anemia. At least in theory, the development of anemia during gefitinib treatment could result from triggering of eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and by cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling potentially stimulating eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i) and generation of oxidative stress. The present study explored, whether gefitinib stimulates eryptosis and, if so, whether its effect involves Ca2+ entry and/or oxidative stress. Methods: Flow cytometry was employed to quantify cell volume from forward scatter, phosphatidylserine exposure at the cell surface from annexin-V-binding, [Ca2+]i from Fluo3-fluorescence, and reactive oxygen species (ROS) abundance from 2’,7’-dichlorodihydrofluorescein diacetate (DCFDA) dependent fluorescence. Results: A 48 hours exposure of human erythrocytes to gefitinib (≥ 2 µg/ml) significantly decreased forward scatter and significantly increased the percentage of annexin-V-binding cells. Gefitinib did not significantly increase Fluo3-fluorescence but the effect of gefitinib on annexin-V-binding was significantly blunted by removal of extracellular Ca2+. Gefitinib further significantly increased DCFDA fluorescence. Conclusions: Gefitinib triggers erythrocyte shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part dependent on extracellular Ca2+ and paralleled by oxidative stress.
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NYQUIST-BATTIE, CYNTHIA, LAURA E. FRANK, DEANNA LUND, and DANIEL V. LIM. "Optimization of a Fluorescence Sandwich Enzyme-Linked Immunosorbent Assay for Detection of Escherichia coli O157:H7 in Apple Juice." Journal of Food Protection 67, no. 12 (December 1, 2004): 2756–59. http://dx.doi.org/10.4315/0362-028x-67.12.2756.
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Sandwich enzyme-linked immunosorbent assay, especially when coupled with biosensor technology, is a simple methodology that can rapidly screen juices for Escherichia coli O157:H7 contamination. However, sampling directly from apple juice and ciders has been postulated to reduce immunoassay sensitivity. In fluorescence sandwich enzyme-linked immunosorbent assays using commercially available polyclonal or monoclonal antibodies, sampling pasteurized apple juice spiked with E. coli O157:H7 compared to spiked phosphate-buffered saline shifted the range of detection. The spiked apple juice range of detection was 104 to 106 CFU/ml, whereas that for spiked phosphate-buffered saline was 106 to 108 CFU/ml, representing a hundredfold difference in sensitivity. Apple juice also increased background fluorescence intensity (P < 0.001) while reducing the net fluorescence intensity per CFU (P < 0.001). The addition of the polymer polyvinylpyrrolidone to apple juice significantly improved assay performance by increasing sensitivity and net fluorescence intensity per CFU and by reducing background fluorescence. Adjusting pH of apple juice from 3.9 to 7.4 improved assay performance but not to the degree seen with phosphate-buffered saline or polyvinylpyrrolidone-treated apple juice samples. The apple juice polyphenol, epicatechin, reduced net fluorescence intensity in a concentration-dependent manner, a change that was reversed by polyvinylpyrrolidone. Taken all together, these results suggest that polyvinylpyrrolidone can improve detection of O157:H7 in juices by reducing the effect of polyphenols on fluorescence sandwich enzyme-linked immunosorbent assay performance.
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Hagimori, Masayori, Mana Taniura, Naoko Mizuyama, Yasushi Karimine, Shigeru Kawakami, Hideo Saji, and Takahiro Mukai. "Synthesis of a Novel Pyrazine–Pyridone Biheteroaryl-Based Fluorescence Sensor and Detection of Endogenous Labile Zinc Ions in Lung Cancer Cells." Sensors 19, no. 9 (May 2, 2019): 2049. http://dx.doi.org/10.3390/s19092049.
Full textAbstract:
A small extent of endogenous labile zinc is involved in many vital physiological roles in living systems. However, its detailed functions have not been fully elucidated. In this study, we developed a novel biheteroaryl-based low molecular weight fluorescent sensor, 3-(phenylsulfonyl)-pyrazine–pyridone (5b), and applied it for the detection of endogenous labile zinc ions from lung cancer cells during apoptosis. The electron-withdrawing property of the sulfonyl group between the phenyl ring as an electron donor and the pyridone ring as a fluorophore inhibited the intramolecular charge transfer state, and the background fluorescence of the sensor was decreased in aqueous media. From the structure–fluorescence relationship analysis of the substituent effects with/without Zn2+, compound 5b acting as a sensor possessed favorable properties, including a longer emission wavelength, a large Stokes shift (over 100 nm), a large fluorescence enhancement in response to Zn2+ under physical conditions, and good cell membrane permeability in living cells. Fluorescence imaging studies of human lung adenocarcinoma cells (A549) undergoing apoptosis revealed that compound 5b could detect endogenous labile zinc ions. These experiments suggested that the low molecular weight compound 5b is a potential fluorescence sensor for Zn2+ toward understanding its functions in living systems.
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Sato, Hidetoshi, Satoshi Wada, and Hideo Tashiro. "Fluorescence Backgroundless Ti: Sapphire Laser Using Acousto-Optical Tunable Filter for Raman Spectroscopic Measurements." Applied Spectroscopy 56, no. 10 (October 2002): 1303–7. http://dx.doi.org/10.1366/000370202760355019.
Full textAbstract:
The background noise inherent to tunable lasers, which emit broad band spontaneous fluorescence from the laser-active medium, is detrimental for sensitive Raman measurement. Using the diffraction effect in an acousto-optic device, we have developed a fluorescence backgroundless Ti: sapphire laser suited for near-infrared Raman spectroscopy. A Raman excitation profile consisting of series of Raman spectra of deoxygenated hemoglobin aqueous solutions was measured by changing excitation wavelengths, revealing the high potential of this laser as a spectroscopic light source.
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Yano, Shuya, Yong Zhang, Fuminari Uehara, Yukihiko Hiroshima, Shinji Miwa, Robert M. Hoffman, and Ming Zhao. "Effect of salmonella typhimurium A1-R on quiescent cancer cells that do not respond to standard chemotherapy." Journal of Clinical Oncology 31, no. 15_suppl (May 20, 2013): e13576-e13576. http://dx.doi.org/10.1200/jco.2013.31.15_suppl.e13576.
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e13576 Background: Quiescent cancer cells are a major impediment to treating solid cancer with chemotherapy, since in most tumors the majority of the cells are quiescent. Methods: The cell-cycle phase of each human MKN45 gastric cancer cell was imaged using a fluorescence ubiquitination cell cycle indicator (FUCCI). With FUCCI, quiescent cancer cells express mKusabira-Orange fluorescent protein (red) and proliferating cells express mAzami-Green fluorescent protein (green). FUCCI-labeled cancer cells in tumor spheres and subcutaneous tumor in nude mice were treated with Salmonella typhimurium A1-R. Results: Time-lapse confocal imaging showed that cancer cells in tumor spheres in serum-free culture become and remained quiescent. S. typhimurium A1-R infected and killed quiescent cancer cells in tumor spheres. In contrast, cytotoxic agents did not kill the quiescent cancer cells in the tumor spheres. S. typhimurium A1-R targeting of FUCCI-expressing subcutaneous tumors growing in nude mice resulted in the killing of quiescent cancer cells resistant to cytotoxic agents. Conclusions: S. typhimurium A1-R can kill quiescent cancer cells which suggests a new therapeutic paradigm potentially more effective than current therapeutics which are ineffective against quiescent cancer cells.
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Mines, C. H., A. Ghadouani, and G. N. Ivey. "Dying to find the source – the quantitative use of rhodamine WT as a proxy for soluble point source pollutants in closed pipe surface drainage networks." Hydrology and Earth System Sciences Discussions 6, no. 3 (June 23, 2009): 4535–62. http://dx.doi.org/10.5194/hessd-6-4535-2009.
Full textAbstract:
Abstract. Rhodamine WT (RWT), a xanthene dye, may serve as a proxy for soluble pollutants within quantitative tracing studies investigating point source contaminant transport. This study quantified the effects of altering the concentration, pH, temperature and salinity of a RWT solution on the detected fluorescence of RWT within the laboratory prior to a field release of RWT within a closed pipe urban drainage network. All RWT solutions exhibited stability and <10% variation from the expected concentration over a thirteen hour laboratory study period; pH related quenching of RWT fluorescence of up to 14.9% was observed for solutions with pH <3.9; and increasing salinity of RWT solution was found to have a negligible quenching effect. In direct contrast to previous studies RWT fluorescence was found to directly correlate with temperature of solution, and a temperature correction factor was determined and tested. If these effects were combined in an additive manner, the maximum potential underestimation and overestimation of RWT concentration are approximately 30% and 20% respectively. The field release study succeeded in detecting RWT at concentrations two orders of magnitude greater than background fluorescence, and RWT peak concentration prediction using longitudinal dispersion theory was achieved within 10% of the observed peaks.
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Leach, Sydney. "CN spectroscopy and cosmic background radiation measurements." Canadian Journal of Chemistry 82, no. 6 (June 1, 2004): 730–39. http://dx.doi.org/10.1139/v04-036.
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A source of possible error, not previously considered, in the determination of the cosmic background radiation (CBR) temperature Tγ(CN) from interstellar CN absorption is proposed. This concerns the intrinsic assumption of the validity of using HönlLondon rotational line intensity factors to determine the rotational components of the oscillator strengths of the R(0), R(1), and P(1) lines of the CN B2Σ+X2Σ+ (0,0) transition. Published data on interstellar CN absorption shows that Tγ(CN) is slightly greater than the standard cosmological CBR temperature, Tγ(COBE) = 2.725 K, determined by the COBE satellite, even when local excitation effects of CN are taken into account. From this difference, an estimation was made of the maximum corrections, a few percent, to the ratio of the line strengths and to the ratio of the HönlLondon factors. Fluorescence lifetime data were shown to give similar values for the corrections as well as providing evidence for intramolecular coupling between the relevant B2Σ+ state rotational levels and close-lying levels of the A2Π state, this being responsible for rendering the standard HönlLondon factors invalid. Key words: CN spectroscopy, cosmic background radiation, HönlLondon factors.
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Bissinger, Rosi, Sabrina Waibel, Ghada Bouguerra, Abdulla Al Mamun Bhuyan, Salem Abbès, and Florian Lang. "Enhanced Eryptosis Following Exposure to Lopinavir." Cellular Physiology and Biochemistry 37, no. 6 (2015): 2486–95. http://dx.doi.org/10.1159/000438601.
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Background/Aims: The protease inhibitor lopinavir, used for the treatment of HIV infections, triggers suicidal death or apoptosis of nucleated cells. Side effects of lopinavir include anemia, which could in theory result from stimulation of suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and by phospholipid scrambling of the cell membrane leading to phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include oxidative stress, increase of cytosolic Ca2+ activity ([Ca2+]i), and ceramide. The present study explored, whether lopinavir induces eryptosis. Methods: Flow cytometry was employed to estimate phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) abundance from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, reduced glutathione (GSH) from mercury orange fluorescence and ceramide abundance utilizing labelled specific antibodies. Hemolysis was estimated from haemoglobin concentration of the supernatant. Results: A 48 hours exposure of human erythrocytes to lopinavir significantly increased the percentage of annexin-V-binding cells (≥ 10 µg/ml), significantly decreased forward scatter (≥15 µg/ml), significantly increased hemolysis (≥ 15 µg/ml), significantly increased Fluo3-fluorescence (20 µg/ml), and significantly increased DCFDA fluorescence (20 µg/ml) but did not significantly modify ceramide abundance. The effect of lopinavir on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+. Conclusion: Lopinavir treatment of erythrocytes from healthy volunteers is followed by cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to stimulation of ROS formation and Ca2+ entry.
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Tanaka, Katsuya, Dorothee Weihrauch, Lynda M. Ludwig, Judy R. Kersten, Paul S. Pagel, and David C. Warltier. "Mitochondrial Adenosine Triphosphate–regulated Potassium Channel Opening Acts as a Trigger for Isoflurane-induced Preconditioning by Generating Reactive Oxygen Species." Anesthesiology 98, no. 4 (April 1, 2003): 935–43. http://dx.doi.org/10.1097/00000542-200304000-00021.
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Background Whether the opening of mitochondrial adenosine triphosphate-regulated potassium (K(ATP)) channels is a trigger or an end effector of anesthetic-induced preconditioning is unknown. We tested the hypothesis that the opening of mitochondrial K(ATP) channels triggers isoflurane-induced preconditioning by generating reactive oxygen species (ROS) in vivo. Methods Pentobarbital-anesthetized rabbits were subjected to a 30-min coronary artery occlusion followed by 3 h reperfusion. Rabbits were randomly assigned to receive a vehicle (0.9% saline) or the selective mitochondrial K(ATP) channel blocker 5-hydroxydecanoate (5-HD) alone 10 min before or immediately after a 30-min exposure to 1.0 minimum alveolar concentration (MAC) isoflurane. In another series of experiments, the fluorescent probe dihydroethidium was used to assess superoxide anion production during administration of 5-HD or the ROS scavengers N-acetylcysteine or N-2-mercaptopropionyl glycine (2-MPG) in the presence or absence of 1.0 MAC isoflurane. Myocardial infarct size and superoxide anion production were measured using triphenyltetrazolium staining and confocal fluorescence microscopy, respectively. Results Isoflurane (P < 0.05) decreased infarct size to 19 +/- 3% (mean +/- SEM) of the left ventricular area at risk as compared to the control (38 +/- 4%). 5-HD administered before but not after isoflurane abolished this beneficial effect (37 +/- 4% as compared to 24 +/- 3%). 5-HD alone had no effect on infarct size (42 +/- 3%). Isoflurane increased fluorescence intensity. Pretreatment with N-acetylcysteine, 2-MPG, or 5-HD before isoflurane abolished increases in fluorescence, but administration of 5-HD after isoflurane only partially attenuated increases in fluorescence produced by the volatile anesthetic agent. Conclusions The results indicate that mitochondrial K(ATP) channel opening acts as a trigger for isoflurane-induced preconditioning by generating ROS in vivo.
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Clarke, Timothy, Michael E. Evans, Graham Hepworth, Peter J. Moate, and John A. Stewart. "Mordant factors that affect the fluorescence and counting of somatic cells by instruments." Journal of Dairy Research 62, no. 3 (August 1995): 373–94. http://dx.doi.org/10.1017/s0022029900031095.
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SummaryThe requirement for accuracy in the counting of somatic cells in milk has been increased by the use of this measurement as a basis of payment for milk. This paper reports on several factors that can substantially affect efficiency of staining and cell detection in various types of fluorescent cell counters. These mordant factors include sample age, chemical preservative, other sample preservation treatments and reagent properties. Experiments were conducted in several laboratories with Fossomatic 215 and 360 cell counting instruments to measure the effect of these mordant factors on both cell fluorescence and cell count. Counting efficiency was as low as 20% for some instruments counting some particular samples, depending upon the combination of adverse mordant factors. The problem of poor counting efficiency was also not detected by calibration samples that had more favourable mordant treatments applied. The adverse mordant factors were shown to reduce the intensity of fluorescence of cells for all instruments. We suggest that a significant reduction in counting efficiency due to these adverse mordant factors is most likely to occur when the particular instrument already has an intrinsic inability to separate cell fluorescence properly from background noise. This paper also discusses quality control samples, the impact of various factors on discriminator curves and the use of appropriate measures to maintain good calibration adjustment.
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Wilton, J. C., G. M. Matthews, R. D. Burgoyne, C. O. Mills, J. K. Chipman, and R. Coleman. "Fluorescent choleretic and cholestatic bile salts take different paths across the hepatocyte: transcytosis of glycolithocholate leads to an extensive redistribution of annexin II." Journal of Cell Biology 127, no. 2 (October 15, 1994): 401–10. http://dx.doi.org/10.1083/jcb.127.2.401.
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We have used fluorescent derivatives of the choleretic bile salts cholate and chenodeoxycholate, the cholestatic salt lithocholate, and the therapeutic agent ursodeoxycholate to visualize distinct routes of transport across the hepatocyte and delivery to the canalicular vacuole of isolated hepatocyte couplets. The cholate and chenodeoxycholate derivatives produced homogeneous intracellular fluorescence and were rapidly transported to the vacuole, while the lithocholate analogue accumulated more slowly in the canalicular vacuole and gave rise to punctate fluorescence within the cell. Fluorescent ursodeoxycholate showed punctate intracellular fluorescence against a high uniform background indicating use of both pathways. Inhibition of vesicular transport by treatment with colchicine and Brefeldin A had no effect on the uptake of any of the compounds used, but it dramatically impaired delivery of both the lithocholate and the ursodeoxycholate derivatives to the canalicular vacuole. We conclude that while the chenodeoxycholate and cholate analogues traverse the hepatocyte by a cytoplasmic route, lithocholate and ursodeoxycholate analogues are transported by vesicle-mediated transcytosis. Treatment of couplets with glycine derivatives of lithocholate and ursodeoxycholate, but not cholate or chenodeoxycholate, led to a marked relocalization of annexin II, which initially became concentrated at the basolateral membrane, then moved to a perinuclear distribution and finally to the apical membrane as the incubation progressed. This suggests that lithocholate and ursodeoxycholate treatment leads to a rapid induction of transcytosis and that annexin II exchange occurs upon membrane fusion at all stages of the hepatocyte transcytotic pathway. These results indicate that isolated hepatocyte couplets may provide an inducible model system for the study of vesicle-mediated transcytosis.
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Stockinger, Katja, Rosi Bissinger, Ghada Bouguerra, Salem Abbès, and Florian Lang. "Enhanced Eryptosis Following Exposure to Carnosic Acid." Cellular Physiology and Biochemistry 37, no. 5 (2015): 1779–91. http://dx.doi.org/10.1159/000438541.
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Background/Aims: The phenolic abietane diterpene component of rosemary and sage, carnosic acid, may either induce or inhibit apoptosis of nucleated cells. The mechanisms involved in the effects of carnosic acid include altered mitochondrial function and gene expression. Human erythrocytes lack mitochondria and nuclei but are nevertheless able to enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Cellular mechanisms involved in the stimulation of eryptosis include oxidative stress, increase of cytosolic Ca2+ activity ([Ca2+]i), and ceramide formation. The present study explored, whether and how carnosic acid induces eryptosis. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCFDA dependent fluorescence and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to carnosic acid significantly increased the percentage of annexin-V-binding cells (2.5 µg/ml), significantly decreased forward scatter (10 µg/ml), significantly increased Fluo3 fluorescence (10 µg/ml), significantly increased ceramide abundance (10 µg/ml), significantly increased hemolysis (10 µg/ml), but significantly decreased DCFDA fluorescence (10 µg/ml). The effect of carnosic acid on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+. Conclusion: Carnosic acid triggers cell shrinkage and phospholipid scrambling of the human erythrocyte cell membrane, an effect paralleled by and/or in part due to Ca2+ entry and increased ceramide abundance.
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Waibel, Sabrina, Rosi Bissinger, Ghada Bouguerra, Salem Abbès, and Florian Lang. "Saquinavir Induced Suicidal Death of Human Erythrocytes." Cellular Physiology and Biochemistry 37, no. 5 (2015): 1973–82. http://dx.doi.org/10.1159/000438558.
Full textAbstract:
Background/Aims: The antiretroviral protease inhibitor saquinavir is used for the treatment of HIV infections. Effects of saquinavir include induction of apoptosis, the suicidal death of nucleated cells. Saquinavir treatment may further lead to anemia. In theory, anemia could result from accelerated erythrocyte loss by enhanced suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include Ca2+ entry with increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress with increase of reactive oxygen species (ROS) and ceramide. The present study explored, whether and how saquinavir induces eryptosis. Methods: To this end, flow cytometry was employed to estimate erythrocyte volume from forward scatter, phosphatidylserine exposure at the cell surface from annexin-V-binding, [Ca2+]i from Fluo3-fluorescence, ROS abundance from DCFDA fluorescence and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to saquinavir significantly decreased forward scatter (≥ 5 µg/ml), significantly increased the percentage of annexin-V-binding cells (≥ 10 µg/ml), significantly increased Fluo3-fluorescence (15 µg/ml), significantly increased DCFDA fluorescence (15 µg/ml), but did not significantly modify ceramide abundance. The effect of saquinavir on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+. Conclusions: Saquinavir triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to stimulation of ROS formation and Ca2+ entry.