Ultimate use of two-photon fluorescence microscopy to map orientational behavior of fluorophores

International audienceThe orientational distribution of fluorophores is an important reporter of the structure and function of their molecular environment. Although this distribution affects the fluorescence signal under polarized-light excitation, its retrieval is limited to a small number of parameters. Because of this limitation, the need for a geometrical model (cone, Gaussian, etc.) to effect such retrieval is often invoked. In this work, using a symmetry decomposition of the distribution function of the fluorescent molecules, we show that polarized two-photon fluorescence based on tunable linear dichroism allows for the retrieval of this distribution with reasonable fidelity and without invoking either an a priori knowledge of the system to be investigated or a geometrical model. We establish the optimal level of detail to which any distribution can be retrieved using this technique. As applied to artificial lipid vesicles and cell membranes, the ability of this method to identify and quantify specific structural properties that complement the more traditional molecular-order information is demonstrated. In particular, we analyze situations that give access to the sharpness of the angular constraint, and to the evidence of an isotropic population of fluorophores within the focal volume encompassing the membrane. Moreover, this technique has the potential to address complex situations such as the distribution of a tethered membrane protein label in an ordered environment

Thioflavine-T and Congo Red reveal the polymorphism of insulin amyloid fibrils when probed by polarization-resolved fluorescence microscopy.

International audienceAmyloid fibrils are protein misfolding structures that involve a β-sheet structure and are associated with the pathologies of various neurodegenerative diseases. Here we show that Thioflavine-T and Congo Red, two major dyes used to image fibrils by fluorescence assays, can provide deep structural information when probed by means of polarization-resolved fluorescence microscopy. Unlike fluorescence anisotropy or fluorescence detected linear dichroism imaging, this technique allows to retrieve simultaneously both mean orientation and orientation dispersion of the dye, used here as a reporter of the fibril structure. We have observed that insulin amyloid fibrils exhibit a homogeneous behavior over the fibrils' length, confirming their structural uniformity. In addition, these results reveal the existence of various structures among the observed fibrils' population, in spite of a similar aspect when imaged with conventional fluorescence microscopy. This optical nondestructive technique opens perspectives for in vivo structural analyses or high throughput screening

Developments in the Photonic Theory of Fluorescence

Conventional fluorescence commonly arises when excited molecules relax to their ground electronic state, and most of the surplus energy dissipates in the form of photon emission. The consolidation and full development of theory based on this concept has paved the way for the discovery of several mechanistic variants that can come into play with the involvement of laser input – most notably the phenomenon of multiphoton-induced fluorescence. However, other effects can become apparent when off-resonant laser input is applied during the lifetime of the initial excited state. Examples include a recently identified scheme for laser-controlled fluorescence. Other systems of interest are those in which fluorescence is emitted from a set of two or more coupled nanoemitters. This chapter develops a quantum theoretical outlook to identify and describe these processes, leading to a discussion of potential applications ranging from all-optical switching to the generation of optical vortices

Intensity Weighted Subtraction Microscopy Approach for Image Contrast and Resolution Enhancement

We propose and demonstrate a novel subtraction microscopy algorithm, exploiting fluorescence emission difference or switching laser mode and their derivatives for image enhancement. The key novelty of the proposed approach lies in the weighted subtraction coefficient, adjusted pixel-by-pixel with respect to the intensity distributions of initial images. This method produces significant resolution enhancement and minimizes image distortions. Our theoretical and experimental studies demonstrate that this approach can be applied to any optical microscopy techniques, including label free and non-linear methods, where common super-resolution techniques cannot be used

Ticagrelor – toward more efficient platelet inhibition and beyond

Michał J Kubisa,1 Mateusz P Jezewski,1 Aleksandra Gasecka,2,3 Jolanta M Siller-Matula,4 Marek Postuła1 1Department of Experimental and Clinical Pharmacology, Centre for Preclinical Research and Technology (CEPT), 21st Chair and Department of Cardiology, Medical University of Warsaw, Warsaw, Poland; 3Vesicle Observation Centre, Laboratory of Experimental Clinical Chemistry, Academic Medical Centre, University of Amsterdam, the Netherlands; 4Department of Cardiology, Medical University of Vienna, Vienna, Austria Abstract: Novel antiplatelet drugs, including ticagrelor, are being successively introduced into the therapy of atherothrombotic conditions due to their superiority over a standard combination of clopidogrel with acetylsalicylic acid in patients with acute coronary syndromes (ACS). A P2Y12 receptor antagonist, ticagrelor, is unique among antiplatelet drugs, because ticagrelor inhibits the platelet P2Y12 receptor in a reversible manner, and because it demonstrates a wide palette of advantageous pleiotropic effects associated with the increased concentration of adenosine. The pleiotropic effects of ticagrelor comprise cardioprotection, restoration of the myocardium after an ischemic event, promotion of the release of anticoagulative factors and, eventually, anti-inflammatory effects. Beyond the advantageous effects, the increased concentration of adenosine is responsible for some of ticagrelor’s adverse effects, including dyspnea and bradycardia. Large-scale clinical trials demonstrated that both standard 12-month therapy and long-term use of ticagrelor reduce the risk of cardiovascular events in patients with ACS, but at the expense of a higher risk of major bleeding. Further trials focused on the use of ticagrelor in conditions other than ACS, including ischemic stroke, peripheral artery disease and status after coronary artery bypass grafting. The results of these trials suggest comparable efficacy and safety of ticagrelor and clopidogrel in extra-coronary indications, but firm conclusions are anticipated from currently ongoing studies. Here, we summarize current evidence on the superiority of ticagrelor over other P2Y12 antagonists in ACS, discuss the mechanism underlying the drug–drug interactions and pleiotropic effects of ticagrelor, and present future perspectives of non-coronary indications for ticagrelor. Keywords: ticagrelor, P2Y12, antiplatelet drugs, myocardial infarction, acute coronary syndromes, pleiotropis

Zmiany składu fenoli i tokoferoli w różach brokułu w trakcie krótkotrwałego przechowywania

Broccoli (Brassica oleracea L. var. italica) contains numerous biologically active compounds. However, their levels change during processing and storage. The objective of the research study was to determine the effect of short-term storage (1 ÷ 4 days) of broccoli heads at different temperatures (3 ºC, 16 ºC, and 21 ºC) on the contents of phenolic acids, selected flavonoids (quercetin and kaempferol), and tocopherols. Broccoli heads were harvested in autumn when they reached their maximum size and their buds were 2 mm in diameter. The content of phenolics and tocopherols depended on the storage time of broccoli heads. The lowest content of phenolics was found immediately after the broccoli was harvested, whereas the highest content thereof was reported after 4 days of storage. However, the storage temperature did not impact the contents of those compounds. In the research study, the content of caffeic acid was reported to be highest in the fresh broccoli heads; the content of sinapic acid was the second-highest, and that of the ferulic acid was the third-highest. The content of kaempferol in the broccoli heads was higher than that of quercetin. No differences were reported in the content of quercetin and kaempferol at particular storage temperatures on every single day of the experiment. An increase was reported only when compared with the control sample (the 0 day of storage). In the broccoli heads, the predominant tocopherol was α-tocopherol. The lowest loss in the total tocopherols occurred while storing at a temperature of 3 ºC and the highest: at a temperature of 21 ºC.Brokuł (Brassica oleracea L. var. italica) zawiera liczne związki biologicznie aktywne. Ich poziom ulega jednak zmianie w trakcie przetwarzania i przechowywania. Przedmiotem badań było określenie wpływu krótkotrwałego przechowywania róż brokułu (1 ÷ 4 dni) w różnych temperaturach (3, 6 i 21 ºC) na zawartość kwasów fenolowych, wybranych flawonoidów (kwercetyny i kemferolu) oraz tokoferoli. Brokuły były zbierane jesienią, kiedy róże osiągnęły swoje maksymalne rozmiary, a ich pączki wynosiły około 2 mm. Zawartość fenoli i tokoferoli zależała od czasu przechowywania róż. Najmniejszą zawartość fenoli stwierdzono tuż po zbiorze, a największą – po 4 dniach przechowywania. Na zawartość tych składników nie miała jednak wpływu temperatura przechowywania. Wśród badanych kwasów fenolowych stwierdzono największą zawartość kwasu kawowego, następnie synapinowego i ferulowego. Zawartość kemferolu w różach brokułu była większa niż zawartość kwercetyny. Nie było różnic pod względem zawartości kemferolu i kwercetyny w poszczególnych temperaturach przechowywania, w każdym dniu badań. Wzrost obserwowano tylko w porównaniu z próbą kontrolną (dzień 0). Dominującym tokoferolem w różach brokułu był α-tokoferol. Najmniejszy ubytek sumy tokoferoli uzyskano podczas przechowywania w temperaturze 3 ºC, a największy – w temperaturze 21 ºC