Yingtai: Key Operating Points of Fluorescence Microscopy

Yingtai: Key Operating Points of Fluorescence Microscopy  

A fluorescence microscope is a precision instrument used to observe fluorescently labeled samples. Its key operating points include light source adjustment, filter selection, sample preparation, and safe usage.  

I. Detailed Operating Steps  

1. Environmental Preparation  

   - Dark Environment: When using a fluorescence microscope, ensure the working environment is as dark as possible to reduce background light interference and enhance the visibility of fluorescent signals.  

   - Adequate Ventilation: Ensure the microscope lamp housing is well-ventilated to prevent overheating, which may affect the stability of the light source.  

2. Light Source Adjustment  

   - Igniting the Mercury Lamp: Insert the power plug of the mercury lamp housing into the socket on the back of the fluorescence power supply box. Then, plug the power supply box into a 220V external power source. Press the start switch to ignite the mercury lamp. If the lamp does not ignite successfully on the first attempt, try multiple times until it stabilizes.  

   - Centering the Lamp: Adjust the horizontal and vertical screws on the lamp housing to center the arc of the mercury lamp. Adjust the reflector to separate the reflected image of the light source from the arc, ensuring uniform illumination.  

3. Filter Selection  

   - Excitation Filter Selection: Select the appropriate excitation filter set based on the characteristics of the fluorescently stained specimen. Ensure its transmission wavelength matches the transmission wavelengths of the dichroic beam splitter and the barrier filter, in accordance with Stokes' law.  

   - Filter Set Numbering: The excitation filter set numbers are usually engraved on the filter wheel. Select the appropriate set accurately according to experimental requirements.  

4. Sample Observation  

   - Objective Lens Selection: Choose a suitable fluorescence objective lens (e.g., 10X, 40X, or 100X) based on the required magnification. For high-magnification observation, add glycerol between the specimen and the objective lens to ensure no bubbles or impurities are present.  

   - Focusing: Adjust the coarse focus knob until the outline of the fluorescent image is visible. Then, use the fine focus knob to refine the focus until the image is clear.  

5. Microphotography

   - Exposure Time: Fluorescent images are often dim, requiring longer exposure times during microphotography. Avoid instrument vibration to ensure image clarity.  

   - Immediate Recording: Fluorescence is prone to fading. Capture images promptly after observation to prevent signal degradation.  

II. Operational Precautions  

1. Safety Measures  

   - Wear Protective Glasses: When adjusting the light source, wear protective glasses to prevent ultraviolet radiation from harming the eyes.  

   - Avoid Arbitrary Operations: Strictly follow the instruction manual. Do not skip steps or change the sequence arbitrarily to prevent operational errors that may damage the equipment or lead to experimental failure.  

2. Equipment Maintenance  

   - Regular Cleaning: After use, turn off the power promptly and cover the microscope with a dust cover to prevent dust from entering the optical system.  

   - Avoid Frequent Switching: The mercury lamp has a limited lifespan. Frequent switching will shorten its service life. It is recommended to turn off the power collectively after experiments.