Final Report Summary - CSRR (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.
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.