Séminaire impromptu - Martin Oheim"Imaging the near-membrane space simultaneously at high spatial and temporal resolution - Why, where we are at and where we are heading to"15 déc. 2016 à 11:30 (Nouveau Bâtiment Neurocampus)
Most structured illumination microscopes use a physical or synthetic grating that is projected into the sample plane to generate a periodic illumination pattern. Albeit simple and cost-effective, this arrangement hampers fast or multi-color acquisition, which is a critical requirement for time-lapse imaging of cellular and sub-cellular dynamics.
We designed and implemented an interferometric approach allowing large-field, fast, dual-color imaging at an isotropic 100-nm resolution based on a sub-diffraction fringe pattern generated by the interference of two colliding evanescent waves. Our all-mirror-based system generates illumination patterns of arbitrary orientation and period, limited only by the illumination aperture (NA) of the objective lens, the response time of a fast, piezo-driven tip-tilt mirror and the available fluorescence signal. At low µW laser powers suitable for long-period observation of life cells and with a camera exposure time of 20 ms, our system permits the acquisition of super-resolved 50 µm by 50 µm images at 3.3 Hz.
The possibility it offers for rapidly adjusting the pattern between images is particularly advantageous for experiments that require multi-scale and multi-color information. We demonstrated the performance of our instrument by imaging the collective dynamics of mitochondria and the endoplasmic reticulum (ER) in cultured cortical astrocytes. As an illustration of dual-color excitation dual-color detection, we also resolve interaction sites between near-membrane mitochondria and the endoplasmic reticulum. Our TIRF-SIM microscope – now housing five lasers spanning the entire visible wavelength range – provides a versatile, compact and cost-effective arrangement for large-field super-resolution imaging, allowing the investigation of co-localization and dynamic interactions between organelles – important questions in both cell biology and neurophysiology
- TIRF (Total Internal Reflection Fluorescence)
Martin Oheim : ELS, in press
- Fluorescent Probes Used for Measuring Intracellular Calcium
Martin Oheim : ELS, 2015
- Astrocyte VAMP3 vesicles undergo Ca2+ -independent cycling and modulate glutamate transporter trafficking.
Li D, Hérault K, Zylbersztejn K, Lauterbach MA, Guillon M, Oheim M, Ropert N : J Physiol,2015
- FRET-Based Nanobiosensors for Imaging Intracellular Ca(2+) and H⁺ Microdomains.
Zamaleeva AI, Despras G, Luccardini C, Collot M, de Waard M, Oheim M, Mallet JM, Feltz A : Sensors (Basel), 2015
- Eliminating unwanted far-field excitation in objective-type TIRF. Part I. identifying sources of nonevanescent excitation light.
Brunstein M, Teremetz M, Hérault K, Tourain C, Oheim M : Biophys J, 2014
- Eliminating unwanted far-field excitation in objective-type TIRF. Part II. combined evanescent-wave excitation and supercritical-angle fluorescence detection improves optical sectioning.