Fluorescence microscopy is an essential tool in bio-imaging, yet there are no widely adopted standards for the calibration of fluorescent microscopes. Calibration provides a wide range of information relating to microscope performance. Without calibration, images taken on two separate microscopes cannot be directly compared as they may have differing magnifications, illumination intensities or detector sensitivities. As the range of microscopy techniques capturing 3D information continues to increase, the need for standardisation becomes ever greater. Widely used methods for determining microscope performance are currently limited to basic techniques such as fluorescent beads, which don’t form a regularly spaced pattern and reflective etched gratings, which are limited to being two-dimensional and require changes to the microscope filter sets. Using ultrafast laser processing inside plastic substrates, we demonstrate the generation of bright fluorescent patterns in three dimensions offering new possibilities for calibration in fluorescence microscopy. The fabricated calibration slides can be used to quantify a range of parameters that determine microscope performance. For example, spatial distortions within the field of view can be quantified by a regular array of bright fluorescent points. Other patterns can determine factors such as detector linearity, field flatness and changes in the point spread function across the field of view and over depth. The patterns can additionally be used to calibrate spatial length-scales and for colour channel registration.