Shining Light on Photobleaching: An Artifact That Causes Unnecessary Excitation Among Pathologists.

Abstract

Photobleaching artifact occurs when fluorescence intensity decreases following light exposure. Slides stained with fluorescent techniques may be stored in the dark until primary diagnostics. Experimental evidence suggesting the rate of photobleaching and necessity of dark storage is lacking. To compare photobleaching rate on direct immunofluorescence and Thioflavin T slides stored in ambient room light conditions and exposed to excitatory wavelengths. During 2 iterations of the experiment, 45 slides were prepared, 42 with immunofluorescent antibodies plus 3 with thioflavin, from skin and kidney biopsies. The experimental group was stored in room light conditions in comparison to the control in the dark, at room temperature. Further, 1 immunofluorescence slide and 1 thioflavin slide were exposed to excitatory fluorescent light for several hours. Significant photobleaching was defined as an integer decrease in score (scale, 0-3). Exposure times ranged from 152 to 3034 hours. Nine of the 42 immunofluorescence slides (21%) photobleached after a minimum exposure of 152 hours to room light, with no significant difference between the experimental and control groups (all P values >.05). The immunofluorescence slide exposed to fluorescent light for 4 hours showed marked photobleaching in the exposed field but not elsewhere. No thioflavin slides showed clinically significant photobleaching under any conditions. Clinically significant photobleaching of slides exposed to room light may occur after a few days, but not a few hours (unless exposed to excitatory fluorescent light). Conversely, thioflavin-stained slides did not photobleach when exposed to ambient room air and photobleached only negligibly when exposed to excitatory fluorescent light.