How Much Do You Know About Fluorescence Microscopy
Fluorescence microscopes generally use high-intensity mercury lamps as excitation light sources. Filters are used to filter out unwanted light, leaving only the high-intensity, pure light that excites the fluorophore. After the monochromatic light irradiates the sample through the objective lens, the sample will be excited to emit light (fluorescence), and both the fluorescence and the excitation light will return along the optical path of the objective lens. In this case, a dichroic mirror is required to filter the excitation light. , letting only the fluorescence we need to see through.
This fluorescence reaches the eyepiece along the light path of the microscope, and then enters our eyes, where we can see the fluorescence emitted by the fluorophore.
Pre-check and adjustment of the fluorescence microscope:
(1) Before each fluorescence observation, it is necessary to routinely check the filament alignment, optical path focus, aperture diaphragm, and field diaphragm settings of the fluorescence device.
(2) Whether the required fluorescence excitation/emission filter assembly has been installed in the converter, whether the fluorescence microscope objective lens is properly configured, and remove the oil stains and dust on the front lens of the objective lens.
(3) If phase contrast observation of transmitted light is carried out at the same time, it is necessary to check the conjugation of the center of the condenser and the phase contrast ring opposite to the objective lens.
(4) Check whether the sample carrier (slide glass, cover glass and other utensils) is covered with liquid or dust, and whether the thickness is within the calibrated working distance range of the objective lens. The sliced sample should not be too thick, preferably ≤10 μm.
(5) Because the lighting source contains ultraviolet rays, a brown light-shielding plate is placed above the front of the stage to prevent ultraviolet rays from damaging the retina.
(6) Voltage instability will reduce the service life of the high-pressure mercury lamp, and the light source power supply is equipped with a voltage stabilizer.
(7) In order to prolong the life of the mercury lamp, it can be turned off 15 minutes after it is turned on; once the fluorescent power of the mercury lamp is turned off, it needs to wait at least 10 minutes to restart the mercury vapor to cool down and return to the original state, otherwise the life of the lamp will be affected.
Image observation by fluorescence microscope:
(1) About 5-10 minutes after turning on the fluorescent light source, the intensity of the excitation light tends to be stable, and the sample is loaded for observation; in order to prevent the fluorescence quenching of the sample caused by excessive excitation light during the process of focusing and looking for objects, first zoom out the fluorescence microscope Adjust the excitation light to a moderate intensity with an aperture diaphragm or add an ND filter, and move the sample stage regularly. After confirming the mirror image, adjust to the fluorescent state for shooting and recording.
(2) Adjustments for poor image quality. In addition to sample preparation factors, the necessary adjustments that can be made are:
① Exclude light-shielding or light-limiting devices in the imaging optical path, such as DIC accessories, ND filters, etc.
②Readjust the receiver focus and aperture diaphragm size of the fluorescence microscope.
③ Carefully adjust the coverage difference correction ring of the fluorescence microscope objective lens.
Application Points of Fluorescence Microscopy
Fluorescence microscopy uses "actinic fluorescence" imaging. If the selected excitation wavelength is in the near-ultraviolet region (320-400nm), which is invisible to the naked eye, the emission spectrum of fluorescence is also shorter than the average wavelength of ordinary light mirror light sources. improve. High-energy photons collide with electrons, causing the electrons to transition from the ground state to the excited state. The electrons in the excited state are very unstable and will fall back to the ground state. In this process, part of the thermal energy will be consumed and new photons will be emitted. The new photon has a lower energy than the original photon and therefore has a longer wavelength. Since the new photon wavelength is different from the photon wavelength of the incident light, the two beams of light with different wavelengths are separated by a certain optical processing method, so that we only see the emitted new photons (fluorescence signal), that is, the fluorescence microscope sees fluorescence images.
