9 March 2015 Quantitative imaging of intact cardiac tissue using remote focusing microscopy
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Remote focussing microscopy offers many advantages when acquiring volumetric data from living tissue. The all-optical means of refocussing does not agitate the specimen by moving either the stage or imaging objective. Aberrationcompensated imaging extends over volumes as large as 450 μm x 450 μm x 200 μm (X, Y and Z) allowing data to be collected from hundreds of cells. The speed with which refocussing can be achieved is limited only by the mechanical movement of a small (2 mm diameter) mirror. Using a pair of oblique imaging planes to rapidly acquire (<200ms) depth information temporally freezes residual tissue motion in the arrested heart.

This paper discusses the progress of remote focussing microscopy from a novel imaging technique to a reliable tool in the life sciences. Specifically, we describe recent efforts to achieve the accurate calibration of both distance and orientation within the imaging volume. Using a laser machined fluorescent specimen it is possible to identify, with high sensitivity, small (<1%) depth-dependent magnification changes which are a linear function of axial misalignment of the imaging objective. The sensitivity of the calibration procedure limits distortion to <1 μm over the entire imaging volume. This work finds direct application in identifying the microscopic effects of chronic disease in the living heart.
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A. D. Corbett, A. D. Corbett, R. A. B. Burton, R. A. B. Burton, G. Bub, G. Bub, T. Wilson, T. Wilson, "Quantitative imaging of intact cardiac tissue using remote focusing microscopy", Proc. SPIE 9330, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXII, 93300R (9 March 2015); doi: 10.1117/12.2076133; https://doi.org/10.1117/12.2076133

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