A workflow for comparison of light microscopes image quality achieved at identical irradiance

This is a preliminary disclosure; protocol under development A biological light microscopy experiment involves a trade-off between its “gain” (i.e. the useful information content of the images) and the associated “costs” of phototoxicity and photobleaching. These effects are largely determined by irradiance (radiant flux (optical power) per unit area) of a given microscope configuration. Increasing irradiance typically improves image quality (metrics like signal-to-noise ratio (SNR)), but at the expense of increased photobleaching and/or phototoxicity. Therefore, a “bio-specimen-aware” comparison of image quality produced by different microscope configurations should be performed under conditions of identical irradiance and exposure time. Here, we propose a workflow that includes measurements of the illuminated area and illumination power to calculate irradiance, adjustment of microscopes under comparison to achieve identical irradiance, and calculation of SNR as a commonly used image quality metric.