Two Common Microscope Observation Methods
1. Dark‑field Observation
Dark‑field observation employs dark‑field illumination. Unlike bright‑field observation, the illuminating light is not viewed directly; only the light reflected or diffracted by the specimen is detected. As a result, the field of view has a dark background while the specimen appears bright.
Dark‑field observation is based on the Tyndall effect in optics. Dust particles are invisible to the human eye under direct intense light due to light diffraction. When light strikes the particles obliquely, light reflection makes the particles appear enlarged and visible.
The special accessory required for dark‑field observation is a dark‑field condenser. It prevents the light beam from passing vertically upward through the specimen. Instead, it redirects light to illuminate the specimen at an oblique angle. The illuminating light does not enter the objective lens directly, and bright images are formed by the reflected and diffracted light from the specimen surface. The resolution of dark‑field observation is far higher than that of bright‑field observation, reaching 0.02–0.004 μm.
2. Phase‑contrast Microscopy
In the development of optical microscopes, the invention of phase‑contrast microscopy represents a major achievement in modern microscopy. The human eye can only distinguish the wavelength (colour) and amplitude (brightness) of light waves. For colourless and transparent biological specimens, light passes through with little change in wavelength or amplitude, making them difficult to observe under bright‑field conditions.
Phase‑contrast microscopy performs inspection based on the optical path difference of the specimen. It effectively uses the interference of light to convert phase differences (invisible to the human eye) into distinguishable amplitude differences. Even colourless transparent substances can be clearly visualized. This greatly facilitates the observation of living cells, so phase‑contrast microscopy is widely used with inverted microscopes.
The basic principle of the phase‑contrast microscope is to convert the optical path difference of visible light transmitted through the specimen into an amplitude difference, thereby improving the contrast between different structures and making them clearly visible. After passing through the specimen, light refracts, deviates from its original path and is retarded by 1/4 of a wavelength. An additional retardation or advance of 1/4 wavelength results in a total optical path difference of 1/2 wavelength. The two beams are superimposed and interfere, increasing or decreasing the amplitude to enhance contrast. Structurally, the phase‑contrast microscope has two special features that distinguish it from ordinary optical microscopes.
