Optical microscopy is a powerful technique that allows visualisation of structures that are invisible with the naked eye. With innovations in computer technology, detectors, optics and photochemistry, the field of microscopy has developed significantly in recent years with highly sophisticated systems commercially available as a matter of routine. With the advent of new microscopes new challenges arise, particularly in the quantification of data so that the end-user may better understand the images obtained. For accurate quantification, the microscope has to be accurately aligned and calibrated, and one must be aware of the tolerances of use. Some of these parameters, such as resolution, are well-documented but other properties of the microscope, such as the effect of the dose of light on the specimen or the size of the optical focus and its repeatability over time, are less well understood. The aim of this thesis is to identify areas which are generally not tested or calibrated by the vendor or end user of an optical microscope, and provide methods that can inform on the performance. Following the introduction, the second and third chapters describe improved techniques for assessing the alignment and performance of a laser scanning microscope. The second part of this thesis, Chapters 4 and 5, describe the implications of accurate measurements in microscopy to measurements in both a life sciences application and a physical sciences application. Chapter 6 concludes the thesis and includes a discussion of future opportunities that may follow.