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High-speed 3D imaging with the oblique plane microscope

le 3 novembre 2016 à 11 h

Vincent Maioli, Photonics Group, Imperial College London, UK


Lieu(x) :        Amphithéatre Poisson, Ecole Polytechnique


Contact :      Emmanuel Beaurepaire
                     emmanuel.beaurepaire at polytechnique.edu


The oblique plane microscope (OPM) is a light-sheet microscope enabling fast acquisition of 3-D volumes for samples ranging from single cells to tumour spheroids. OPM uses a single high numerical aperture for both light sheet illumination and fluorescence collection and it can therefore be implemented as add-on to a standard microscope frame. Hence OPM is compatible with conventional sample preparation techniques such as microscope slides or standard multi-well plates. The current microscope acquires in two emission channels simultaneously enabling e.g. two separate fluorescent probes to be imaged or ratiometric FRET imaging.

 

We will demonstrate the capabilities of OPM to image fast processes by imaging cardiomyocytes to study the relationship between calcium sparks characteristics and cellular structures (t-tubules). From our high-speed 3D data acquired at 25 volumes per second, we have shown that in cells from hearts with myocardial infarction, calcium waves predominantly originate from well-tubulated regions of the cell. Associated data show that calcium sparks occur most frequently in well-tubulated regions and are larger and longer between two t-tubules.

 

A high-speed 3-D imaging plate-reader based on the OPM will then be presented. It enables three-dimensional multi-channel acquisition based on mechanical translation of the sample through the light-sheet, with the camera acquisition synchronized to the displacement of the stage. This approach readily scales to imaging samples prepared in multi-well plates and to time-lapse studies. We demonstrate its application to 3-D imaging of cells growing in collagen in a 96-well plate and time-lapse movies of tumour spheroids. Also, spatio-temporal glucose dynamics have been observed over several hours using a glucose FRET biosensor in live spheroids seeded in a 96-well plate.


À télécharger :  Séminaire MAIOLI