Abstract
Remote focussing microscopy allows sharp, in-focus images to be acquired at high speed from outside of the focal plane of an objective lens without any agitation of the specimen. However, without careful optical alignment, the advantages of remote focussing microscopy could be compromised by the introduction of depth-dependent scaling artifacts. To achieve an ideal alignment in a point-scanning remote focussing microscope, the lateral (XY) scan mirror pair must be imaged onto the back focal plane of both the reference and imaging objectives, in a telecentric arrangement. However, for many commercial objective lenses, it can be difficult to accurately locate the position of the back focal plane. This paper investigates the impact of this limitation on the fidelity of three-dimensional data sets of living cardiac tissue, specifically the introduction of distortions. These distortions limit the accuracy of sarcomere measurements taken directly from raw volumetric data. The origin of the distortion is first identified through simulation of a remote focussing microscope. Using a novel three-dimensional calibration specimen it was then possible to quantify experimentally the size of the distortion as a function of objective misalignment. Finally, by first approximating and then compensating the distortion in imaging data from whole heart rodent studies, the variance of sarcomere length (SL) measurements was reduced by almost 50%.
Highlights
Remote focussing microscopy is a technique that enables optically sectioned, in-focus images of a specimen to be taken outside of the focal plane of the imaging objective
This is the ideal alignment configuration as rays passing through the center of the physical aperture are steered by the lens to arrive normal to the focal plane
By simulating the lateral and axial displacements of the imaging objective, it is possible to predict the impact of the displacements on the imaging volume
Summary
Remote focussing microscopy is a technique that enables optically sectioned, in-focus images of a specimen to be taken outside of the focal plane of the imaging objective. The remote focussing technique explored in this paper uses a reference objective to axially displace the focal spot (Botcherby et al, 2007, 2008a,b; Salter et al, 2009). In conventional microscopes, illuminating the objective with defocussed light to illuminate points outside of the focal plane generates strong spherical aberrations, resulting in a blurry image. In a remote focussing microscope, these aberrations are pre-compensated using a matched “reference” objective. This is a significant development as it allows images of the entire specimen volume to be acquired
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