IMAGINDNA · Advanced DNA imaging: improving spatial resolution and contrast through photoswitching
7РП — „Хора“ (Действия „Мария Кюри“)
- Период
- 2013-02-01 → 2017-01-31
- Финансиране от ЕС
- 100 000 €
- Участници
- 1
- Схема
- MC-CIG
Линиите свързват координатора с партньорите.
Накратко на български
Методите за визуализиране на ДНК се подобряват чрез комбиниране на два вида микроскопия, за да се видят хромозоми и наноматериали в детайли. Това помага за по-точното определяне на структурата и свойствата на сложни биологични и химични обекти в наномащаб.
Кратко обяснение, генерирано от езиков модел по текста на CORDIS. Оригиналът е по-долу.
Резултати накратко
Advanced DNA imaging: improving spatial resolution and contrast through photoswitching
The general aim of this proposal is to improve the methodology for advanced imaging of DNA using different and complementary approaches, and to use the improved methods in the investigation of chromosomes and other complex structures at the nanoscale. To this end, a novel microscope in which is possible to correlate atomic force microscopy (AFM) and super-resolution fluorescence imaging has been implemented. A protocol for sample preparation that is compatible with both techniques has been developed and applied to proof-of-principle experiments with fluorescently labeled DNA. This novel methodology can be used to benchmark novel super-resolution imaging methods, as well as to obtain complementary information about the structure and properties of (bio)materials. The latter has been exemplified on a hybrid nanomaterial that consists of protein fibrils functionalized with organic fluorophores and quantum dots. Our experiments allow combining information about the topography and number of filaments that compose a fibril, as well as the emissive properties and nanoscale spatial distribution of the attached fluorophores. This technique offers great potential for the characterization of multifunctionalized hybrid materials, a key challenge in nanoscience. Progress in fluorescent labeling of DNA has also been achieved by understanding and exploiting the properties of CyDNA, a biopolymer consisting in DNA labeled in high density with cyanine dyes. The fluorescence quenching mechanisms have been elucidated, and the knowledge has allowed redesigning CyDNA to improve its properties. CyDNA has also been re-engineered into a reversible fluorescence photoswitchable biopolymer, and constitutes a new class of photoactive DNA-based nanomaterial that is of great interest for advanced microscopy applications. We have exploited fluorescence photoswitching in CyDNA to perform proof-of-principle super-resolution and optical lock-in detection (OLID) imaging. The grant has greatly contributed to the consolidation of the fellow and her group at the host institution, and to the establishment of new national and international collaborations.
Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз
Цел на проекта
Fluorescence microscopy is one of the most convenient and widespread tools used in the life sciences. An important challenge, however, is to improve its spatial resolution, which is limited to about 200 nm. Recent “super-resolution” techniques such as photoactivation-localization microscopy (PALM) can provide images with a spatial resolution of tens of nm. Most studies performed with these techniques have imaged the nanoscale distribution of proteins. However, little progress has been seen on DNA super-resolution imaging due to challenges in labelling. My main research is aimed at exploring new ways to label DNA in high density with photoswitchable fluorophores and improving spatial resolution in fluorescence microscopy. This will allow opening up new opportunities to study a broad range of problems in Biology and Nanoscience. My recent work has shown that PALM-like imaging of DNA can achieve a spatial resolution below 40 nm by using intercalating cyanine dyes in combination with a buffer that promotes photoblinking. This proposal aims at optimizing this approach, and at finding new alternatives for DNA super-resolution imaging. I propose two research lines: 1) Use correlative atomic force microscopy and PALM-like imaging to optimize the above methodology. As first targets, DNA origami will be used. Correlative microscopy will then be applied to study chromosome structure; 2) Study the photophysical properties at the ensemble and single-molecule level of a new material, CyDNA (DNA highly substituted with cyanine dyes in a controllable way). Bringing together Cy3 and Cy5 dyes in the same CyDNA with high density results in a photoswitch with new properties. CyDNA photoswitching will be used for super-resolution imaging in combination with fluorescence in situ hybridization, and also to improve image contrast with optical lock-in detection imaging. Further opportunities to apply the developed methodology will also be identified throughout the fellowship
Оригинален текст от CORDIS (на английски).
Участници
- FUNDACION IMDEA NANOCIENCIA · MadridКоординаторИспания
Връзки
Данни: CORDIS, © Европейски съюз