The group led by FNP Prize winner Prof. Daniel T. Gryko from the Institute of Organic Chemistry of the Polish Academy of Sciences in Warsaw has developed innovative dyes for use in fluorescence microscopy, emitting very strong red light, which makes them potentially suitable for studying deeper biological structures. The scientists also managed to explain why these compounds emit light despite the presence of nitro groups in their structure, which was previously thought to suppress fluorescence. The results were published by the prestigious journal Angewandte Chemie. The research was financed by the Foundation for Polish Science under the TEAM programme, from European funds within the framework of the Smart Growth Operational Programme.
?Fluorescence is the ability to emit light of a specific colour following excitation with light of a specific wavelength. Fluorescent compounds are highly valued and widely used. Fluorescent pens are a highly popular example known to everyone. Also gaining popularity are tablets, laptops and even television sets with displays made of OLEDs, which are diodes based on organic compounds emitting red, green and blue light. Fluorescent compounds are also used in modern molecular biology and medical diagnostics. They are used for observing, with fluorescence microscopes, various cell organelles and proteins, and also to track cellular processes,? said Prof. Daniel Gryko.
Fluorescence microscopy has a great advantage over conventional optical microscopy in terms of resolution. Due to the wavelike nature of light, an optical microscope does not allow imaging structures smaller than about 200 nanometres. Electron microscopes provide orders of magnitude better resolution, but only allow the examination of inanimate objects, which must be placed in a vacuum and bombarded by an electron beam. Electron microscopy cannot be used for studying living organisms and the natural processes occurring in them. This is where fluorescence microscopy steps in. To make an observation using a fluorescence microscope, it is necessary to have fluorescent molecules, or fluorophores, which are called dyes or markers. They are attached to the object that is to be revealed under the microscope, such as a specific protein, which makes it possible to observe, e.g. specific antibodies or proteins involved in the development of neurodegenerative diseases, such as Parkinson?s, Alzheimer?s and Huntington?s. The most advanced technique in fluorescence microscopy is stimulated emission depletion (STED), which, in addition to the beam of excitation light, uses an additional beam that depletes fluorescence on the edges of the excited point. This yields a super-resolution image. The Nobel Prize was awarded for the development of STED fluorescence microscopy in 2014. STED made it possible to study the interactions between proteins in cells and tissue differentiation in embryonic development with unprecedented precision.
Fluorescent dyes intended for use in STED microscopy must feature, in addition to fluorescence, a number of other significant properties, such as the ability to penetrate through the cell membranes of living cells, and a high photostability. The latter property is often highly problematic, as many compounds disintegrate under a high-power laser beam, which is used in fluorescence microscopes. ?Designing and then synthesising enhanced dyes will allow to further the development of STED microscopy and facilitate its future use in medical diagnostics,? said Prof. Daniel Gryko, who has managed to achieve just that. The group working under his supervision, in cooperation with scientists from France and Germany, has developed a new class of durable fluorescent tags ? next generation Diketopyrrolopyrroles ? characterised by very intensive red light emission. ?Intensive red light emission opens up an opportunity to use these organic compounds as fluorescent probes, as this is the most visible colour under a fluorescence microscope,? emphasised Prof. Gryko. This achievement is described in the article ?How to Make Nitroaromatics Glow: Next Generation Large, ??Shaped, Centrosymmetric Diketopyrrolopyrroles,? which was published in June of this year in one of the most renowned scientific journals, Angewandte Chemie. Its lead author is the winner of the START programme of the Foundation for Polish Science, Dr. Kamil Skonieczny. The publication also explains why Diketopyrrolopyrroles are strongly fluorescent despite having two nitro groups in their structure. Previously scientists assumed that the presence of a nitro group almost always suppresses fluorescence, so compounds which contain it cannot be used as fluorescent tags, with one exception. The researches demonstrated that when specific conditions are met, a nitro group does not affect the compound?s fluorescence, which is important, because its presence also improves the tag?s stability. It should be added that this discovery is patent pending.
New tags were synthesised and the fluorescence mechanism of these compounds was explained as part of the scientific research conducted under the TEAM 3/2016 grant financed by the Foundation for Polish Science from the funds of the European Regional Development Fund within the framework of the Smart Growth Operational Programme.
Prof. Daniel Gryko is the Director of the Institute of Organic Chemistry of the Polish Academy of Sciences. His main research interests include the chemistry of functional dyes, especially corroles, which are macrocyclic dyes with a variety of applications. He is the winner of the MASTER academic grant for professors of the Foundation for Polish Science (2013). He also won grants under the TEAM FNP programme twice. In 2017 he was awarded the Prize of the Foundation for Polish Science, which is considered the most important Polish scientific award.
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Photo: Prof. Daniel Gryko_Photo: Magdalena Wiśniewska-Krasińska/FNP archive