Operating Instructions and Precautions for Fluorescence Microscope
Fluorescence images observed under a fluorescence microscope feature morphological characteristics as well as fluorescence color and brightness. Both factors must be combined for comprehensive result evaluation. Result recording relies on subjective indicators based on visual observation by operators, which is generally reliable for routine qualitative observation. With advances in technology, objective instruments such as microspectrophotometers and image analyzers are increasingly used for documentation and evaluation. Nevertheless, results obtained from these instruments still require subjective interpretation.
Photography with a fluorescence microscope is essential for documenting fluorescence images. Since fluorescence fades rapidly, results must be photographed promptly. The procedure is largely the same as conventional photomicrography, except that high-speed photographic films of ASA 200 or higher are required. Ultraviolet light causes severe fluorescence photobleaching. For example, FITC-labeled specimens lose 50% of their fluorescence brightness after 30 seconds of UV irradiation. Therefore, slow exposure will fail to capture fluorescence images. Most research-grade fluorescence microscopes are equipped with semi-automatic or fully automatic photomicrography systems.
Precautions
① Operate strictly in accordance with the manufacturer's manual of the fluorescence microscope; do not alter operating procedures arbitrarily. ② Observations should be carried out in a darkroom. After entering the darkroom, switch on the power and ignite the high-pressure mercury lamp for 5–15 minutes. Start specimen observation only after the light source stabilizes and your eyes adapt fully to darkness.
③ Protect your eyes from ultraviolet radiation. Wear protective goggles when adjusting the light source.
④ Each observation session is recommended to last 1–2 hours. The luminous intensity of the ultra-high-pressure mercury lamp gradually declines after 90 minutes, and fluorescence weakens accordingly. Fluorescence of specimens also fades noticeably after 3–5 minutes of UV exposure. The maximum continuous observation time shall not exceed 2–3 hours.
⑤ The service life of the fluorescence microscope light source is limited. Examine specimens in batches to save time and prolong lamp life. Use an electric fan for heat dissipation in hot environments. Record the operating time from the very beginning after lamp replacement. If the lamp goes out, wait until it cools completely before reignition. Avoid frequent restarting of the lamp.
⑥ Observe stained specimens immediately after staining, as fluorescence will fade over time. Specimens can be stored at 4°C in polyethylene plastic bags to slow fluorescence decay and prevent evaporation of the mounting medium.
⑦ Grading standard for fluorescence intensity: four grades are generally adopted: "−": No fluorescence or only faint fluorescence detectable "+": Barely detectable but definite fluorescence signal "++": Bright fluorescence visible
