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Shining Light on Photobleaching: An Artifact That Causes Unnecessary Excitation Among Pathologists
Benzion Samueli1, Yarden Kezerle1, Jacob Dreiher1
1From the Department of Pathology (Samueli, Kezerle, Osipov) and Hospital Administration (Dreiher), Soroka University Medical Center, Be'er Sheva, Israel; Medical School of International Health and Faculty of Health Sciences (Samueli, Kezerle, Dreiher, Steckbeck) and the Department of Chemistry (Baraban), Ben Gurion University of the Negev, Be'er Sheva, Israel; and Institute of Pathology, E. Wolfson Medical Center, Holon, Israel, and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel (Vaknine).
Context.—:
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.
Objective.—:
To compare photobleaching rate on direct immunofluorescence and Thioflavin T slides stored in ambient room light conditions and exposed to excitatory wavelengths.
Design.—:
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).
Results.—:
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.
Conclusions.—:
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.
Insights
Photobleaching artifact in immunofluorescence slides can occur after days in room light, but not hours. Thioflavin T slides show minimal photobleaching, suggesting dark storage may not always be necessary.
Area of Science:
- Histopathology
- Biomedical Imaging
- Fluorescence Microscopy
Background:
- Photobleaching artifact reduces fluorescence intensity after light exposure.
- Slides stained with fluorescent techniques are often stored in the dark.
- Evidence on photobleaching rates and dark storage necessity is limited.
Purpose of the Study:
- To compare photobleaching rates of direct immunofluorescence and Thioflavin T slides.
- To assess photobleaching under ambient room light and excitatory wavelengths.
- To determine the necessity of dark storage for stained slides.
Main Methods:
- 45 slides (42 immunofluorescence, 3 Thioflavin T) from skin/kidney biopsies were used.
- Slides were stored in room light (experimental) or dark (control).
- Specific slides underwent prolonged exposure to excitatory fluorescent light.
Main Results:
- 21% of immunofluorescence slides showed photobleaching after >=152 hours in room light (P >.05 vs. dark).
- Immunofluorescence slides exposed to fluorescent light for 4 hours showed marked photobleaching.
- No Thioflavin T slides exhibited clinically significant photobleaching under any condition.
Conclusions:
- Clinically significant photobleaching of immunofluorescence slides may occur after days in room light, not hours (unless exposed to excitatory light).
- Thioflavin T slides are resistant to photobleaching in ambient air and show negligible photobleaching under excitatory light.
- Dark storage may not be universally required, especially for Thioflavin T-stained slides.
