CSRR · Correlative Super Resolution and Real-Time Imaging of Herpes Virus Infection
FP7 — People (Marie Curie Actions)
- Duration
- 2011-03-01 → 2015-02-28
- EU contribution
- €100,000
- Participants
- 1
- Scheme
- MC-IRG
Lines connect the coordinator with its partners.
Results in brief
Correlative Super Resolution and Real-Time Imaging of Herpes Virus Infection
Fluorescence microscopy is a key enabling technique for biology. In the last decade, one of the biggest challenges in far-field fluorescence microscopy, namely the diffraction limit of spatial resolution, has been overcome. New superresolution imaging techniques that provide nanometer-scale spatial resolution have been developed and these new advances have brought us into the era of nanoscopy. While these techniques have found several novel applications in biology, one of their major limitations has been low temporal resolution. Spatial and temporal resolution must often be balanced against each other in superresolution nanoscopy, making it difficult to study highly dynamic processes with the needed spatiotemporal resolution. The goal of this project is to circumvent this limitation by developing an all-optical correlative imaging technique that combines the high temporal resolution of real-time live cell imaging and single particle tracking with the high spatial resolution of superresolution nanoscopy. The workflow of this all-optical correlative imaging method is as follows: A live-cell movie of a biological process of interest is recorded with high temporal resolution, the sample is then fixed in situ on the microscope stage and immunostained with antibodies in order to record a superresolution image of a target of interest, which can then be precisely aligned and correlated with the live cell movie. This new method allows researchers to interpret their data in a new light, putting dynamic information into the context of ultrastructural information at high spatiotemporal resolution. In the course of the project we have successfully: 1. Implemented the correlative live-cell and super-resolution imaging method by developing protocols for on-stage in situ sample fixation, labeling and algorithms for precise image registration. 2. Automated the correlative live-cell and super-resolution imaging method through the use of microfluidic devices, which has allowed us to streamline the sample preparation and improve throughput. 3. Applied the correlative live-cell and super-resolution imaging method to important biological problems, such as studying the impact of cellular roadblocks on cargo transport and studying correlations between mitochondria dynamics and morphology. 4. Increased the information content of the correlative live-cell and super-resolution imaging method by developing ways to extract precise quantitative information from super-resolution images. Overall, the project has allowed the development of novel imaging technologies expanding the repertoire of available single molecule techniques and opening the doors for applying these tools to several other biological questions. Therefore, it is expected to make a big impact in microscopy, biophysics and biology communities.
Data: CORDIS, © European Union
Project objective
Fluorescence imaging is a powerful technique that has transformed our understanding of biology. Recently, a number of techniques have been developed that overcome one of the fundamental limitations of fluorescence imaging, namely the diffraction limit. With these techniques, it is now possible to resolve sub-cellular architecture in multiple colors and 3D with unprecedented detail. However, the main limitation of these techniques has been the slow acquisition times making it difficult to study dynamic processes. Since biological samples are inherently highly dynamic, this limitation is a major hurdle that needs to be overcome. I will develop a correlative fluorescence imaging technique that combines the capabilities of super resolution and real-time imaging. With this correlative technique it will be possible to observe the dynamics of a biological sample in real-time and subsequently “freeze” the dynamics (by fixation or low temperature) at a time of interest to obtain a super resolution image. The dynamics can therefore be correlated with ultrastructural information, combining the capabilities of real-time and super resolution imaging. I will apply this correlative imaging technique to study infection mechanism of Herpes Simplex Virus (HSV). HSV is a medically important virus that infects neurons and epithelial cells. Besides the health hazards that it poses, HSV also has important implications in gene therapy. However, the details of HSV infection mechanism remain poorly understood. Since HSV infection involves dynamic interactions between virus particles and sub-cellular components, both of which are tens of nanometers in length scale, this is an ideal model system in which the correlative super resolution and real-time imaging technique will lead to important insights that were previously unattainable.""
Original text from CORDIS.
Participants
- FUNDACIO INSTITUT DE CIENCIES FOTONIQUES · CastelldefelsCoordinatorSpain
Links
Data: CORDIS, © European Union