Photodetector offset influences glomerular albumin permeability values dramatically. The offset or “black level” settings determines the lower detection limits on the photomuliplier tube (PMT) detectors found in many laser scanning confocal and multi-photon microscopes. A: effect on detector sensitivity using 2 different offsets on a range of background subtracted Texas red albumin (TRA) solutions in vitro. The average intensity readings from the images were divided by the concentration of TRA to give a direct unit of sensitivity. This unit should be constant across the concentration range if sensitivity is unaffected, as shown when offset is set to 38. Using a higher numerical offset of 45, which forces background values closer to zero, markedly decreases the microscope's ability to correctly detect fluorescence in identical dishes. With detector sensitivity decreased particularly at lower TRA concentrations, the sensitivity value is no longer constant across the range for offset 45. B: list of background subtracted, glomerular sieving coefficients for albumin (GSCA) collected at different offsets for the same set of glomeruli. Note the rapidly decreasing GSCA seen starting at offset 42. There is a 262-fold decrease in GSCA in a comparison of offsets 38 and 52. C and D: identical images of a glomerulus after infusion of TRA, taken at offsets 38 and 45, respectively. The blue color seen in Bowman's space in D (offset 45) indicates the values located therein have a value of zero before background subtraction. Colors alerting the user to pixels in the image having intensity values above or below the detection limit of the detectors are standard in every microscope system (typically red for saturated values and blue or green for values at zero). The GSC for TRA in these experiments, where only the offset was changed for the image, averaged 0.015 ± 0.0047 using offset 38 and 0.00114 ± 0.00096 using offset 45. Values in this, and all of our studies, were background subtracted, with a reference image taken at the corresponding offsets.