Abstract
Light sheet fluorescence microscopy has previously been demonstrated on a commercially available inverted fluorescence microscope frame using the method of oblique plane microscopy (OPM). In this paper, OPM is adapted to allow time-lapse 3-D imaging of 3-D biological cultures in commercially available glass-bottomed 96-well plates using a stage-scanning OPM approach (ssOPM). Time-lapse 3-D imaging of multicellular spheroids expressing a glucose Förster resonance energy transfer (FRET) biosensor is demonstrated in 16 fields of view with image acquisition at 10 minute intervals. As a proof-of-principle, the ssOPM system is also used to acquire a dose response curve with the concentration of glucose in the culture medium being varied across 42 wells of a 96-well plate with the whole acquisition taking 9 min. The 3-D image data enable the FRET ratio to be measured as a function of distance from the surface of the spheroid. Overall, the results demonstrate the capability of the OPM system to measure spatio-temporal changes in FRET ratio in 3-D in multicellular spheroids over time in a multi-well plate format.
Highlights
Speed of these single point scanning approaches are limited by the maximum peak excitation power that can be tolerated by the sample in terms of photobleaching and, in the case of live cells, phototoxicity
In order to deliver the excitation light and collect the fluorescence at 90° using a conventional inverted fluorescence microscope objective and frame, we have adapted the technique of oblique plane microscopy (OPM)[27] so that it can be used for 3-D imaging via a stage-scanning approach, which we refer to as stage-scanning OPM
OPM can be considered as a conventional light sheet microscope setup consisting of perpendicular illumination and detection arms where the light sheet is imaged into a remote sample and the resulting fluorescence is returned using a high numerical aperture image relay designed to achieve equal lateral and axial magnifications
Summary
Speed of these single point scanning approaches are limited by the maximum peak excitation power that can be tolerated by the sample in terms of photobleaching and, in the case of live cells, phototoxicity. Light sheet fluorescence microscopy in conventional multi-well plate arrays has been demonstrated by the use of a fluid filled prism to enable illumination and imaging of the sample at 45° to the plate normal[24] This is an elegant approach, the need for an objective with a working distance that is sufficiently long to allow space for the fluid-filled prism limits the NA of the illumination and collection objectives that can be employed. Advantages of the full 3-D imaging provided by ssOPM include the ability to calculate the minimum distance to the surface of a spheroid for every voxel, rather than relying on assumptions typically required in the analysis of 2-D images of the spheroid’s sphericity and exact selection of the mid-plane when imaging We illustrate these advantages by measuring FRET ratio as a function of distance from surface of spheroid for 16 spheroids in a single time-lapse experiment taking 4 hours across 4 different experimental conditions
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