A three-dimensional (3D) microscope for patterned substrate measurement can include an objective lens, a reflected illuminator, a transmitted illuminator, a focusing adjustment device, an optical sensor, and a processor. The focusing adjustment device can automatically adjust the objective lens focus at a plurality of Z steps. The optical sensor can be capable of acquiring images at each of these Z steps. The processor can control the reflected illuminator, the transmitted illuminator, the focusing adjustment device, and the optical sensor. The processor can be configured to capture first and second images at multiple Z steps, the first image with the pattern using the reflected illuminator and the second image without the pattern using one of the reflected illuminator and the transmitted illuminator.