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Summary NAD(P)H fluorescence emission spectra are recorded from single living cells, by a recently developed multichannel microspectrofluorometric technique, in correlation with the intracellular microelectrophoretic addition of substrate (i. e., glucose-6-P). These spectra may be used as a reference basis in establishing the critical parameters to be followed when the same studies are extended to a variety of cells, submitted to various drug effects or physical treatments. The sum-spectra corresponding to channel by channel (wavelength by wavelength) summation of spectra obtained from various cells within a series, before and after addition of substrate, and their difference spectrum may be normalized and evaluated comparatively. The NAD(P)H emission maximum prior to addition of substrate seems to present a mixture of dehydrogenase-bound and free coenzyme. There is a suggestion that immediately after substrate, i. e., at 5 sec, an increase in free NADH is first observed. While the overall changes in fluorescence intensity associated with substrate are quite large (50–150% increase), the counts (i. e., an expression of photons) associated with shifts in the emission maximum (free vs. bound NAD(P)H changes) are at times barely above noise. Rapid microspectrofluorometry provides in principle the most direct approach for the identification of coenzyme bound to various dehydrogenases in single living cells, but further improvements in spectral resolution and signal-to-noise ratio are required, for a better definition of the spectral shifts which may be observed.
Zusammenfassung Mit Hilfe einer kürzlich entwickelten Mehrkanal-Mikrospektrofluorometer-Methode wurden von einzelnen lebenden Zellen nach intrazellulär mikroelektrophoretischer Substratzugabe (z. B. Glucose-6-P) NAD(P)H Fluoreszenz-Emissionsspektren aufgenommen. Diese Spektren können als Vergleichsbasis bei der Festsetzung der entscheidenden Parameter verwendet werden, wenn die gleichen Untersuchungen auf eine Reihe von Zellen ausgedehnt werden sollen, die verschiedenen Medikamenteffekten oder physikalischer Behandlung ausgesetzt werden. Die Summenspektren, die der kanalmäßigen (wellenlängenmäßigen) Summierung der Spektren von verschiedenen Zellen innerhalb einer Serie, vor und nach Substratzugabe entsprechen, sowie ihre Differenzspektren können normalisiert und vergleichsweise ausgewertet werden. Das NAD(P)H-Emissionsmaximum vor der Substratzugabe scheint ein Gemisch von freiem und dehydrogenasegebundenem Coenzym darzustellen. Unmittelbar nach Substratzugabe (d. h. nach 5 sec) ist ein Anstieg an freiem NADH das erste Mal zu beobachten. Während die mit dem Substrat einhergehenden Gesamtveränderungen der Fluoreszenzintensität recht groß sind (50–150% Anstieg), sind die Impulse (als Effekt der Photonen), die mit einer Verschiebung im Emissionsmaximum verbunden sind (Veränderungen von freiem und gebundenem NAD(P)H) zu manchen Zeiten kaum höher als das Rauschen. Die rasche Mikrospektrofluorometrie stellt im Prinzip die direkteste Methode zur Identifizierung von Coenzymen dar, die an verschiedenen Dehydrogenasen in einzelnen lebenden Zellen gebunden sind. Weitere Verbesserungen der Spektralauflösung und der Empfindlichkeit (signal-to-noise ratio) sind notwendig, um die Spektralverschiebungen, die beobachtet werden, besser auswerten zu können.相似文献
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R. Bruder D. L''Hermite A. Semerok L. Salmon V. Detalle 《Spectrochimica Acta Part B: Atomic Spectroscopy》2007,62(12):1590-1596
During Laser-Induced Breakdown Spectroscopy (LIBS) analysis of white lead pigment (basic lead carbonate, 2PbCO3·Pb(OH)2), used in wall paintings of historical interest, a yellow–brown discoloration has been observed around the crater. This phenomenon faded after a few days exposure under ambient atmosphere. It was established that the mechanism of this discoloration consists in lead oxides (PbO) formation. It was verified by further experiments under argon atmosphere that recombination of lead with oxygen in the plasma plume produces the oxides, which settle around the crater and induce this discoloration. The impact of discoloration on the artwork's aesthetic aspect and the role of atmosphere on discoloration attenuation are discussed. The mechanism is studied on three other pigments (malachite, Prussian blue and ultramarine blue) and threshold for discoloration occurrence is estimated. 相似文献
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John P. Priestle Jr Richard H. Rhyne Jr Jessie B. Salmon Marvin L. Hackerit 《Photochemistry and photobiology》1982,35(6):827-834
Abstract— Trimeric and hexameric solution forms of C-phycocyanin (CPC) from the cyanophyte Agme-nellum quadruplicatum have been isolated and their spectral properties compared to those obtained from single crystals. Although the absorbance peak of a suspension of small C-phycocyanin crystals is red-shifted only 7 nm relative to the solution forms, the single crystal fluorescence is red-shifted 60 nm relative to the solution forms. The crystal fluorescence spectrum exhibits a single peak at LDmax= 708 nm when excited at 514.5 or 530.9 nm and two peaks (LDmax= 661 and 708 nm) when excitation occurs at 568.2 nm. Fluorescence depolarization measurements indicate that extensive energy transfer could occur for both solution and crystal forms with the latter being dependent upon the relative orientation of the crystal with respect to the excitation dipole. Similar results were obtained with B-phycoerythrin (BPE) from the red alga Porphyridium cruentum where the single crystal fluorescence is red-shifted =50nm relative to the solution spectra with two peaks (LDmax= 583 and 617 nm) observed whose relative intensities are dependent on the excitation wavelength (LDmax– 514.5 and 530.9 nm). Single crystal fluorescent lifetimes exhibited considerable shortening relative to that observed for the solution forms. The implications of these results are discussed with respect to the possible relationships of the crystalline structures to the assembly forms present within phycobilisomes. 相似文献
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Alves GA Amato S Anjos JC Appel JA Astorga J Bracker SB Cremaldi LM Darling CL Dixon RL Errede D Fenker HC Gay C Green DR Halling AM Jedicke R Karchin PE Kwan S Leuking LH Mantsch PM de Mello Neto JR Metheny J Milburn RH de Miranda JM da Motta Filho H Napier A Passmore D Rafatian A dos Reis AC Ross WR Santoro AF Sheaff M Souza MH Spalding WJ Stoughton C Streetman ME Summers DJ Takach SF Wallace A Wu Z 《Physical review D: Particles and fields》1994,49(9):R4317-R4320
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Alves GA Amato S Anjos JC Appel JA Astorga J Bracker SB Cremaldi LM Dagenhart WD Darling CL Dixon RL Errede D Fenker HC Gay C Green DR Jedicke R Karchin PE Kennedy C Kwan S Lueking LH de Mello Neto JR Metheny J Milburn RH de Miranda JM da Motta Filho H Napier A Passmore D Rafatian A dos Reis AC Ross WR Santoro AF Sheaff M Souza MH Spalding WJ Stoughton C Streetman ME Summers DJ Takach SF Wallace A Wu Z 《Physical review letters》1996,77(12):2388-2391
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Alves GA Amato S Anjos JC Appel JA Bracker SB Cremaldi LM Darling CL Dixon RL Errede D Fenker HC Gay C Green DR Jedicke R Kaplan D Karchin PE Kwan S Leedom I Lueking LH Luste GJ Mantsch PM de Mello Neto JR Metheny J Milburn RH de Miranda JM da Motta Filho H Napier A Rafatian A dos Reis AC Reucroft S Ross WR Santoro AF Sheaff M Souza MH Spalding WJ Stoughton C Streetman ME Summers DJ Takach SF Wu Z 《Physical review letters》1993,70(6):722-725