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Proceedings Paper

Three-dimensional fluorescence imaging by stage-scanning oblique plane microscopy (Conference Presentation)
Author(s): Vincent Maioli; Frederik Görlitz; Sean Warren; Sunil Kumar; Paul M. W. French; George Chennell; Alessandro Sardini; David Carling; Frederike Alwes; Christopher W. Dunsby

Paper Abstract

Oblique plane microscopy (OPM) is a light-sheet fluorescence microscopy technique that is implemented on a standard inverted microscope frame. OPM uses a single high numerical aperture microscope objective to both produce a tilted excitation light-sheet and to image the fluorescence emitted from the tilted plane back to the cameras. It is therefore compatible with conventional sample-mounting techniques such as microscope slides and multiwell plates. Four excitation laser lines and two high-speed sCMOS cameras with separate emission filters enable the simultaneous imaging of several fluorophores and spectral ratiometric FRET acquisitions. Previously, 3-D OPM imaging has been implemented by remote refocusing. Here, a stage-scanning approach to 3-D OPM imaging is demonstrated - enabling three-dimensional multi-channel acquisition including of multiwell plates - and the synchronization of the stage movement and camera acquisition will be described. The ability of the stage-scanning system to image fields of view larger than the field of view of the primary microscope objective is demonstrated using fluorescently labelled limbs of crustaceans and its ability to perform time-lapse 3-D imaging over 12 hours is demonstrated using a sample of tumor spheroids with an acquisition time of 3 s for a typical spheroid providing 400x1280x1024 voxels per spheroid. We also apply the system to spectral ratiometric Förster resonant energy transfer (FRET) measurements in tumor spheroids expressing a FRET biosensor and in a 96-well plate seeded with cell samples expressing varying concentrations of a FRETting and non-FRETting constructs.

Paper Details

Date Published: 27 April 2016
PDF: 1 pages
Proc. SPIE 9713, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXIII, 971318 (27 April 2016); doi: 10.1117/12.2208808
Show Author Affiliations
Vincent Maioli, Imperial College London (United Kingdom)
Frederik Görlitz, Imperial College London (United Kingdom)
Sean Warren, Imperial College London (United Kingdom)
Sunil Kumar, Imperial College London (United Kingdom)
Paul M. W. French, Imperial College London (United Kingdom)
George Chennell, Imperial College London (United Kingdom)
Alessandro Sardini, Imperial College London (United Kingdom)
David Carling, Imperial College London (United Kingdom)
Frederike Alwes, Ecole Normale Supérieure de Lyon (France)
Christopher W. Dunsby, Imperial College London (United Kingdom)


Published in SPIE Proceedings Vol. 9713:
Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXIII
Thomas G. Brown; Carol J. Cogswell; Tony Wilson, Editor(s)

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