Simple, non-mechanical and automatic calibration approach for axial-scanning microscopy with an electrically tunable lens.

We describe a simple and robust calibration approach for axial-scanning microscopy that realizes axial focus shifts with an electrically tunable lens (ETL). We demonstrate the calibration approach based on a microscope with an ETL placed close to the rear stop of the objective lens. By introducing a target-consisted of repeating lines at one known frequency and placed at a ~45° angle to the imaging path, the calibration method captures multiple images at different ETL currents and calibrates the dependence of the axial focus shift on the ETL current by evaluating the sharpness of the captured images. It calibrates the dependence of the magnification of the microscope on the ETL current by measuring the period of the repeating lines in the captured images. The experimental results show that different from the conventional calibration procedure, the proposed scheme does not involve any mechanical scanning and can simultaneously calibrate the dependence of the axial focus shift and the magnification on the ETL current. This might facilitate imaging studies that require the measurement of fine structures in a 3D volume. We also show the calibration procedure can be used to estimate the radius of a conner-arc sample, fabricated using laser micromachining. We believe that this easy-to-use calibration approach may facilitate use of ETLs for a variety of imaging platforms. It may also provide new insights for the development of novel 3D surface measurement methods. RESEARCH HIGHLIGHTS: The proposed calibration scheme does not involve any mechanical scanning and can simultaneously calibrate the dependence of the axial focus shift and the magnification on the electrically tunable lens (ETL) current. It might facilitate imaging studies that require the measurement of fine structures in a 3D volume, and the use of ETLs for a variety of imaging platforms. It may also provide new insights for the development of novel 3D surface measurement methods.