Fluorescence microscopes have become ubiquitous in life sciences laboratories, including those focused on pharmaceu¬ticals, diagnosis, and forensics. For the past few years, the need for both per¬formance guarantees and quantifiable results has driven development in this area. However, the lack of appropri¬ate standards and reference materials makes it difficult or impossible to com¬pare the results of two fluorescence mi¬croscopes, or to measure performance fluctuations of one microscope over time. Therefore, the operation of fluo¬rescence microscopes is not monitored as often as their use warrants - an is¬sue that is recognized by both systems manufacturers and national metrolo¬gy institutes.
We have developed a new process that enables the etching of long-term stable fluorescent patterns with sub-micrometer sizes in three dimensions inside glass. In this paper, we present, based on this new process, a fluorescent multi-dimensional ruler and a dedicated software that are suitable for monitoring and quality management of fluorescence-based imaging systems (wide-field, confocal, multiphoton, high content machines). In addition to fluorescence, the same patterns exhibit bright- and dark-field contrast, DIC, and phase contrast, which make them also relevant to monitor these types of microscopes.
Non-exhaustively, this new solution enables the measurement of: The stage repositioning accuracy; The illumination and detection homogeneities; The field flatness; The detectors’ characteristics; The lateral and axial spatial resolutions; The spectral response (spectrum, intensity and lifetime) of the system. Thanks to the stability of the patterns, microscope performance assessment can be carried out as well in a daily basis as in the long term.