High speed sCMOS-based oblique plane microscopy applied to the study of calcium dynamics in cardiac myocytes

Abstract

Oblique plane microscopy (OPM) is a form of light sheet microscopy that uses a single high numerical aperture microscope objective for both fluorescence excitation and collection. In this paper, measurements of the relative collection efficiency of OPM are presented. An OPM system incorporating two sCMOS cameras is then introduced that enables single isolated cardiac myocytes to be studied continuously for 22 seconds in two dimensions at 667 frames per second with 960 × 200 pixels and for 30 seconds with 960 × 200 × 20 voxels at 25 volumes per second. In both cases OPM is able to record in two spectral channels, enabling intracellular calcium to be studied via the probe Fluo‐4 AM simultaneously with the sarcolemma and transverse tubule network via the membrane dye Cellmask Orange. The OPM system was then applied to determine the spatial origin of spontaneous calcium waves for the first time and to measure the cell transverse tubule structure at their point of origin. Further results are presented to demonstrate that the OPM system can also be used to study calcium spark parameters depending on their relationship to the transverse tubule structure. Permissions: Wiley-VCH Verlag & Co.KGaA

Time-lapse 3-D measurements of a glucose biosensor in multicellular spheroids by light sheet fluorescence microscopy in commercial 96-well plates

“…1, has been described previously272829. Briefly, light from four excitation sources at different wavelengths was combined using dichroic mirrors and controlled using an acousto-optic tunable filter (AOTF) before being coupled into a single-mode polarisation maintaining optical fibre.…”

“…A dichroic beamsplitter DC (Chroma T510lprxtxt-UF3) and emission filters EM1 (Semrock FF01 550/49) and EM2 (Semrock FF01 482/25) separate the emitted fluorescence into two spectral bands for detection of donor and acceptor fluorescence emission respectively. Specifications for all of the optical components, together with a characterisation of the spatial resolution and optical performance of the OPM system can be found in reference29.…”

“…10 ms later, 2 ms of 515 nm excitation and simultaneous acquisition on camera 1 provided an image of the directly excited acceptor emission. The use of 2 ms camera exposure times means that the sample moves 0.2 μm during the integration, which is less than the measured 0.5 μm in-OPM plane spatial resolution29. The details of the laser power used for each experiment are given in Supplementary Table S2.…”

Fast Objective Coupled Planar Illumination Microscopy

“…Classic LSFM variants such as Selective Plane Illumination Microscopy 4 (SPIM) and OCPI microscopy 6 are limited more severely by the volume scanning bottleneck than the camera frame rate bottleneck. Many newer techniques dispense with the volume scanning bottleneck, but we show that relative to OCPI and SPIM all of these designs compromise either photon efficiency 8,10,15,17,22,23,[26][27][28] or spatial resolution 10,15,17,19,27,29 . As a consequence, no current method is capable of scanning volumes hundreds of microns on a side without compromising optical quality.…”

Imaging subcellular dynamics with fast and light-efficient volumetrically parallelized microscopy

“…To mitigate these demands, several methods have been developed that operate in the absence of objective scanning. These include extended depth of focus, either through the introduction of spherical aberrations [7] or a cubic phase mask [8], oblique illumination [9, 10], confocally-aligned oblique illumination [11], rapidly defocusing the detected wavefront with an electrically tunable lens [12, 13], or remote focusing through wavefront engineering [14]. However, these methods typically sacrifice sensitivity (e.g., due to aberrations, incomplete use of the detection numerical aperture, and inevitably small pixel dwell times) and spatial resolution (usually on the order of a few microns), rendering them incompatible with imaging subcellular structures.…”