Dynamic Imaging  


  HEADProf Dr SHORTE, Spencer / pfid@pasteur.fr
  MEMBERS MEMBERS: Dr. DANCKAERT Anne / Dr. DRAGAVON Joe / Mr. MACHU Christophe / Mme. NICOLAS Marie-Anne / Mme. PERRET Emmanuelle / Dr. ROGERS Kelly / Mr. ROUX Pascal


  Annual Report

The PFID is an applied-sciences group at the Pasteur Institute. Our research is multi-disciplined and collaborative, focused precisely on developing techniques and analysis using “dynamic-imaging” to understand the processes of cell/tissue-biology, and their usurpation by infection and disease.

Research & Development: A definition of dynamic-imaging in Biological Sciences: In basic, and pre-clinical biological sciences the term “Dynamic Imaging” has come to refer to a diverse array of key-technologies that employ the properties of light (particularly fluorescence and bioluminescence) to probe molecular and cellular biology in a quantitative manner through space and time. As such dynamic-imaging is a truly multi- disciplinary field of biological sciences that draws on expertise and knowledge in biochemistry, molecular biology, (bio)-physics, mathematics, cell-biology and informatics alike. Therefore the PFID manages its relatively small team around these competencies. In the context of the Institut Pasteur, where these combined expertise are well harbored and nurtured around cellular microbiology. Arguably an emerging discipline, cellular microbiology is founded upon the basis that experimental approaches in cell biology leverage our understanding in microbiology. Evidently, imaging (& microscopy) is therein a fundamental tool in cell biology and microbiology, and the PFID’s R&D mission is founded upon this methodological and experimental premise.

Our primary R&D aim is to develop novel imaging methods that bring new understanding of host-pathogen interactions at a tissue, cellular and molecular level. In this context, we aim to satisfy the growing need for novel techniques allowing to probe infection, and extrapolate quantitative information on spatio-temporal dynamics in situ. On the other hand we are also motivated to push the limits of existing approaches aiming to enhance their performance thereby broadening their experimental utility. To these ends fluorescent and bioluminescent light based dynamic-imaging techniques offer a powerful tool, and the state-of-the-art is defined by paradigms using so-called multi-dimensional (multi-D, or high-content) dynamic imaging microscopy. Multi-D imaging aims to visualize (acquire) biological events through time and space, and more specifically refers to combinations of: three (3D, volume), four (4D, time), and five (5D, multi-wavelength) dimensional recordings. Today, such data types are grouped under the term high-content data. During the last four years the PFID has been especially interested in the development of in situ high-content imaging techniques and their application to cell biology, cellular microbiology, and microbiology wherein most all our work is focused.

The PFID has three main areas of interest for its R&D activities:

1. DEVELOPMENT AND APPLICATION OF NEW MOLECULAR PROBES- High-content imaging depends upon the use of molecules that yield light signals that may be detected and thereby give information concerning the biology under study. An ideal probe is, therefore, one that yields specific and predictable signals that may be precisely interpreted. There already exist an enormous variety of inorganic, and organic light emitting probes that may be targeted by virtue of their (bio)-chemical and/or molecular specificity. However, the requisite for adaptation of existing probes and development of new probes is never-ending in the face of new questions and the growing complexity of the processes to be measured, and especially the need to monitor multiple processes. The PFID is therefore committed to development of new molecular probes, and new applications of existing probes.

2. DEVELOPMENT OF NOVEL IMAGING MODALITIES, AND BIOLOGICAL MODEL SYSTEMS- High-content imaging requires complex and sophisticated hardware enabling light emitted from samples under study to be recorded in multiple dimensions. In this context spatial and temporal resolution is critical making it important to innovate and implement new instrumentations able to facilitate improved spatial resolution (including super-resolution methods), automated high-speed acquisition (including high-content & high-throughput approaches), and improved read-outs (especially, photon counting & bioluminescence methods, and fluorescence lifetime methods). Currently, we are especially interested in methods that allow to measure, and/or reduce phototoxic effects (where light causes damage to the cells, or tissues under study). Towards these ends the PFID is fully engaged in programs aimed to develop new imaging modalities, and instrumentation, state-of-the-art high-speed detection, super-resolution and automated devices (high-throuput).

3. DEVELOPMENT OF IMAGE ANALYSIS, DATABASE & VISUALIZATION SOLUTIONS- Multi-D imaging is highly dependent upon the use of signal-processing algorithms and software that allow quantitative: a) image-processing, b) light-reconstruction, and c) image-analysis. Ideally, quantitative image-processing applications should allow batch handling of datasets with minimal user intervention, in a fully automated manner, which is simple to apply and reproducible (a problem sometimes referred to as “data pipelining”). The PFID uses a variety of commercially available, and open-source professional imaging software for signal processing, quantitative analysis, and three- dimensional reconstruction. We are also interested in novel bio-informatics approaches to handle statistical problems arising in biological imaging including stochastic paradigms. We are also working on open source software for image database management, for example OME (OMERO) comprising a software suite that takes into consideration image storage, manipulation, metadata structuring, indexing and shared ontology use. A powerful part of OMERO is based on its utility for facilitating scientific communications across WAN/LAN networks based upon client-based sharing image database content using the OMERO suite. The PFID is therefore dedicated to development of informatics and bio-informatics utilities.

Keywords: Dynamic imaging, bioluminescence, fluorescence, high-content, image database



  Publications

Renaud, O., Vina, J. Yong, Y. Machu, C., Trouvé, A., Van der Voort, H., Chalmond, B. and Shorte, S. (2008). High-resolution imaging of living cells in flow suspension using axial-tomography : 3D imaging flow cytometry, Biotechnology J. 3, 53-62

Amino, R., Thiberge, S., Blazquez, S., Baldacci, P., Renaud, O., Shorte, S. and Menard, R. (2007). Imaging malaria sporozoites in the dermis of the mammalian host. Nature Protocols 2, 1705-12.

Arhel, N., A. Genovesio, K. A. Kim, S. Miko, E. Perret, J. C. Olivo-Marin, S. Shorte, and P. Charneau. (2006). Quantitative four-dimensional tracking of cytoplasmic and nuclear HIV-1 complexes. Nature Methods 3:817-24.

Sáez-Cirión, A., Nicola, M-.A., Pancino, G., Shorte, S.L., (2006). Quantitative real-time analysis of HIV-1 gene expression dynamics in single living primary cells. Biotechnol.J. 1 :682-689 Couverture.

Amino R, Thiberge S, Martin B, Celli S, Shorte S, Frischknecht F, Menard R. (2006) Quantitative imaging of Plasmodium transmission from mosquito to mammal. Nature Medicine, 12:220-224.





Activity Reports 2009 - Institut Pasteur
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