ConfocalCheck - A Software Tool for the Automated Monitoring of Confocal Microscope Performance

Abstract

Laser scanning confocal microscopy has become an invaluable tool in biomedical research but regular quality testing is vital to maintain the system’s performance for diagnostic and research purposes.Although many methods have been devised over the years to characterise specific aspects of a confocal microscope like measuring the optical point spread function or the field illumination, only very few analysis tools are available. Our aim was to develop a comprehensive quality assurance framework ranging from image acquisition to automated analysis and documentation. We created standardised test data to assess the performance of the lasers, the objective lenses and other key components required for optimum confocal operation.The ConfocalCheck software presented here analyses the data fully automatically. It creates numerous visual outputs indicating potential issues requiring further investigation. By storing results in a web browser compatible file format the software greatly simplifies record keeping allowing the operator to quickly compare old and new data and to spot developing trends.We demonstrate that the systematic monitoring of confocal performance is essential in a core facility environment and how the quantitative measurements obtained can be used for the detailed characterisation of system components as well as for comparisons across multiple instruments.

Advanced light microscopy core facilities: Balancing service, science and career

“…over time. Several image analysis tools and macros for evaluating performance tests and managing the corresponding data are available (MetroloJ/Fiji, (Hng and Dormann, 2013), PSFj (Theer et al, 2014)). Ideally, the standardized performance test procedures should be designed in collaboration with engineers from the manufacturing companies.…”

3D super-resolution microscopy performance and quantitative analysis assessment using DNA-PAINT and DNA origami test samples

“…In general, in microscopy it is increasingly appreciated that robust test procedures to evaluate system performance are essential since both alignment issues and component performance can degrade instrument performance below design specification [10][11][12][13]. At least of equal importance, such performance checks enable the comparison of data obtained in different laboratories with greater confidence.…”

Using the NoiSee workflow to measure signal-to-noise ratios of confocal microscopes

“…Assessing SNR as part of a general monitoring routine together with measurements of laser intensity and point spread functions (PSF) is therefore important but has been a tedious task so far, as previously described methods to address SNR lack ease of use 4,5 . Some useful tools such as ConfocalCheck help to monitor confocal performances 6 . But whereas the whole purpose is globally the same, ConfocalCheck gives results spanning from laser stability, objective chromatic aberrations, to galvo stability, but does not check detectors performance and SNR.…”