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© 2015 SPIE. 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. Aberration compensated imaging extends over volumes as large as 450 μm × 450 μm × 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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