| 1. |
- Tian, Yuxi, et al.
(författare)
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Quantitative measurement of fluorescence brightness of single molecules
- 2014
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Ingår i: Methods and Applications in Fluorescence. - : IOP Publishing. - 2050-6120. ; 2:3
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Tidskriftsartikel (refereegranskat)abstract
- Single-molecule fluorescence spectroscopy and imaging probe many characteristics of the fluorescence from individual molecules like relative intensity, polarization, lifetime and spectrum. However, such an important and fundamental parameter as absolute fluorescence intensity (or in other words fluorescence brightness), which is proportional to the absorption cross section and fluorescence quantum yield, has not yet been sufficiently exploited in the field. One reason for that is the difficulty of absolute fluorescence brightness measurements. In the present work a detailed description of fluorescence brightness measurements of single molecules is given. We discuss several important factors like the power density and polarization of excitation light, the substrates and the local environment. It is shown that the fluorescence brightness of a single molecule indeed can be measured with sufficient accuracy and used as a powerful parameter for characterization of materials at single molecule/particle level. The brightness of a single object can give similar information as the fluorescence quantum yield that is crucial for understanding the photophysical properties for individual multi-chromophoric systems in inhomogeneous environments.
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| 2. |
- Demirbay, Baris, et al.
(författare)
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Photo-physical characterization of high triplet yield brominated fluoresceins by transient state (TRAST) spectroscopy
- 2023
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Ingår i: Methods and applications in fluorescence. - : IOP Publishing. - 2050-6120. ; 11:4
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Tidskriftsartikel (refereegranskat)abstract
- Photo-induced dark transient states of fluorophores can pose a problem in fluorescence spectroscopy. However, their typically long lifetimes also make them highly environment sensitive, suggesting fluorophores with prominent dark-state formation yields to be used as microenvironmental sensors in bio-molecular spectroscopy and imaging. In this work, we analyzed the singlet-triplet transitions of fluorescein and three synthesized carboxy-fluorescein derivatives, with one, two or four bromines linked to the anthracence backbone. Using transient state (TRAST) spectroscopy, we found a prominent internal heavy atom (IHA) enhancement of the intersystem crossing (ISC) rates upon bromination, inferred by density functional theory calculations to take place via a higher triplet state, followed by relaxation to the lowest triplet state. A corresponding external heavy atom (EHA) enhancement was found upon adding potassium iodide (KI). Notably, increased KI concentrations still resulted in lowered triplet state buildup in the brominated fluorophores, due to relatively lower enhancements in ISC, than in the triplet decay. Together with an antioxidative effect on the fluorophores, adding KI thus generated a fluorescence enhancement of the brominated fluorophores. By TRAST measurements, analyzing the average fluorescence intensity of fluorescent molecules subject to a systematically varied excitation modulation, dark state transitions within very high triplet yield (>90%) fluorophores can be directly analyzed under biologically relevant conditions. These measurements, not possible by other techniques such as fluorescence correlation spectroscopy, opens for bio-sensing applications based on high triplet yield fluorophores, and for characterization of high triplet yield photodynamic therapy agents, and how they are influenced by IHA and EHA effects.
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| 3. |
- Guala, Dimitri, et al.
(författare)
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Experimental validation of predicted cancer genes using FRET
- 2018
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Ingår i: METHODS AND APPLICATIONS IN FLUORESCENCE. - : IOP PUBLISHING LTD. - 2050-6120. ; 6:3
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Tidskriftsartikel (refereegranskat)abstract
- Huge amounts of data are generated in genome wide experiments, designed to investigate diseases with complex genetic causes. Follow up of all potential leads produced by such experiments is currently cost prohibitive and time consuming. Gene prioritization tools alleviate these constraints by directing further experimental efforts towards the most promising candidate targets. Recently a gene prioritization tool called MaxLink was shown to outperform other widely used state-of-the-art prioritization tools in a large scale in silico benchmark. An experimental validation of predictions made by MaxLink has however been lacking. In this study we used Fluorescence Resonance Energy Transfer, an established experimental technique for detection of protein-protein interactions, to validate potential cancer genes predicted by MaxLink. Our results provide confidence in the use of MaxLink for selection of new targets in the battle with polygenic diseases.
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| 4. |
- Ro, Jong Jin, et al.
(författare)
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Fluorescence properties of 6-aryl-2 '-deoxy-furanouridine and pyrrolocytidine and their derivatives
- 2018
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Ingår i: Methods and Applications in Fluorescence. - : IOP Publishing. - 2050-6120. ; 6:1
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Tidskriftsartikel (refereegranskat)abstract
- 2'-deoxyfuranouridine derivatives presenting various aryl groups have been synthesized through Cu(I)-catalyzed intramolecular cyclizations. Moreover, corresponding pyrrolo-dC derivatives have been synthesized and both families of compounds thoroughly characterized using UV/vis and fluorescence spectroscopy as well as time-dependent density functional theory calculations. The photophysical characterization, show that our newly synthesized derivatives of the important pyrrolo-dC family have high fluorescence quantum yields (QYs) and brightness values. Pyrrolo-dC derivative, 3a, shows an environment sensitive QY of up to >60% and brightness of almost 3000, in low polarity solvents and excitation and emission maxima between 365-381 nm and 479-510 nm, respectively, in solvents of different polarities. Two other derivatives, 3b and 3c, show high QYs and brightness values of up to 3300 that are fairly insensitive to their microenvironment. These promising photophysical features suggest future applicability as fluorescent nucleobase analogs.
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| 5. |
- Venugopal Srambickal, Chinmaya, et al.
(författare)
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Cumulative effects of photobleaching in volumetric STED imaging-artefacts and possible benefits
- 2021
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Ingår i: Methods and Applications in Fluorescence. - : Institute of Physics (IOP). - 2050-6120. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- In stimulated emission depletion (STED) imaging, the excitation and depletion laser beams extend well beyond the focal plane in the imaged sample. We investigated how photobleaching resulting from this irradiation can affect STED images, by acquiring 3D images of fluorescent polystyrene beads using a 2D STED microscope, and applying different Z pixel sizes, scanning speeds, resulting in different laser light doses. While higher STED beam irradiances can increase the spatial resolution, they can also significantly increase photobleaching and thereby reduce signal-to-background levels. In 2D STED imaging, based on a single scan within the focal plane, scan parameters can often be selected to avoid photobleaching effects. Upon 3D optical sectioning experiments however, using the same scan parameters, additional cumulative effects of photobleaching may appear, due to the extension of the excitation and depletion laser beams beyond the focal planes being scanned. Apart from a reduction in signal-to-background levels, such photobleaching can lead to an apparent shift of the axial localization of the objects in the images, but also to an increased resolution in the axial dimension. These findings, confirmed by simulations based on a simplified model for photobleaching, suggests some caution in volumetric STED imaging experiments, but also a possibility for enhanced axial resolution in such experiments.
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| 6. |
- Hevekerl, Heike, et al.
(författare)
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Determination of molecular stoichiometry without reference samples by analyzing fluorescence blinking with and without excitation synchronization
- 2015
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Ingår i: METHODS AND APPLICATIONS IN FLUORESCENCE. - : IOP Publishing. - 2050-6120. ; 3:2
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Tidskriftsartikel (refereegranskat)abstract
- Stoichiometry of molecular complexes plays a crucial role in biology. Moreover, for quantitative fluorescence studies, it is often useful to know the number of fluorophores labeled onto the molecules studied. In this work, we propose an approach to determine the number of independent fluorescence emitters on fluorescent molecules based on fluorescence blinking caused by photo-induced triplet state formation, photo-isomerization or charge transfer. The fluorescence blinking is measured under two different excitation regimes, on the same setup, and in one and the same sample. By comparing the fluorescence fluctuations under continuous excitation using Fluorescence Correlation Spectroscopy (FCS), when all the fluorophores are blinking independently of each other, with those occurring under square-pulsed excitation using Transient State (TRAST) spectroscopy, when all fluorophores are blinking in a synchronized manner, the number of fluorophores per molecule can be determined. No calibration sample is needed and the approach is independent of experimental conditions and of the specific environment of the molecules under study. The approach was experimentally validated by labeling double stranded DNA (dsDNA) with different concentrations of the intercalating dye YOYO-1 Iodide. The sample was then measured consecutively by TRAST and FCS and the number of fluorophores per molecule was calculated. The determined numbers were found to agree well with the number of fluorophores per dsDNA, as determined from FCS measurements using additional calibration samples.
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