Introduction
phase difference microscope is the Netherlands Scientist Zernike In 1935, the microscope used to observe the unsatisfinded specimen. Live cells and unsatisfactory biological specimens, due to the difference in refractive index and thickness of the micro-microstructure of the cells, the wavelength and amplitude do not change, only the phase changes (X phase difference), this phase difference Unable to observe. The phase difference microscope is changed by changing this phase difference and utilizing light diffraction and interference phenomenon, and the phase difference is changed to the amplitude difference to observe the living cell and the undefected specimen. The difference between the phase difference microscope and the ordinary microscope is to replace the variable phenket with a cyclic aperture, instead of the common objective lens with an objective lens with a phase plate, and a telescope for a joint.
Theoretical basis
phase difference refers to a difference in the phase of the same light through a medium having different refractive index changes and produces. The phase refers to the position of the fluctuation of light at a certain time. Generally, since the phase difference that can be produced is too small due to the test object (such as the non-colored cell), it is difficult to distinguish, only after the difference is the amplitude (clear dark difference), it can be distinguished. The phase difference determines the difference between the refractive index of the optical wave through the medium and its thickness, or equal to the difference in the product of the refractive index and the thickness (i.e., the difference between the optical path). The phase difference microscope is a mirror test using the optical path of the test.
Cell biology research instruments are differentiated microscopy, transmission electron microscopy and scanning tunnel electron microscopy design or invented Nobel Prize.
Difference
The phase difference microscope has four special structures: phase difference mirror, a turntable concentrator with an annular aperture, a telescope and a green filter. The green filter effect is: reduce the wavelength range of illumination light, reducing the phase change caused by different wavelengths of illumination light.
Uses
observe unsubstated specimens and living cells.
Basic principle
The difference between the optical path difference of different parts of the object is converted into an amplitude (light intensity) by using the difference between the refractive index and thickness of the object of the different structural components of the object. The observed microscope is achieved by a concentrating mirror with a cyclic aperture and a phase difference lens with a phase sheet. Mainly used to observe living cells or non-staple tissue sections, sometimes it can also be used to observe the lack of contrast dyeing samples.
turns the optical path difference of the visible light through the specimen into ambilation, thereby increasing the contrast between various structures, making various structures clearly visible. After the light passes through the specimen, it is delayed from the original optical path, and it is delayed by 1/4 λ (wavelength). If it is increased or decreased by 1/4 λ, the optical path difference becomes 1/2 λ, and the two beam photos are retrofit. Strengthen, the amplitude is increased or reduced, and the contrast is improved. In the configuration, the phase difference microscope is different from 4 specials of ordinary optical microscopy:
1. Annular diaphragm is located between the light source and the concentrator, and the action is to make through the spotlight The light of the device forms a hollow cone, coke to the specimen.
2. Phase plate (Annular Phaseplate) The phase plate coated with magnesium fluoride can be delayed by phase 1/4 λ in the phase plate coated with magnesium fluoride, which can delay the phase of direct light or diffracted light. Divided into two:
(1) a + phase plate: delayed direct shooting 1/4 λ, the optical wave after the optical wave shaft is added, the amplitude is increased, the specimen structure is more bright than the surrounding medium, Form a bright and negative contrast (or negative contrast).
(2) B + phase: Delayed diffraction light 1 / 4λ, the optical wave is subtracted after the two sets of light joint shaft, the amplitude is small, forming a dark reflection (or a positive contrast), the structure is more than the surrounding medium More darker.
3. Coaxial adjustment telescope: The image for adjusting the cyclic aperture is completely matched with the phase plate conjugate surface.
Repair Maintenance
Several problems in the use of differentiated microscopy:
(1) The phase fell when n'n gets the bright and dark contrast, The phase reversal. When the phase difference δ = 0 is not recognized, as the increase in the increase in the increase of δ changes, the phase reversal will occur when δ continues to increase to a certain value. When a 90% high-absorbing value (high contrast) objective lens, this transition is about 0.55λ, which is about 0.33 λ when using a 70% standard light absorbing value. The objective lens of higher absorbing values should be used to distinguish small optical paths.
(2) The halo and the progressive effect are darkened during the imaging of the phase microscope, and the loss is not light due to the delay of the phase, but is not light loss, but the light is refurbished on the image plane. The result of allocation. Therefore, the light in the dark area disappears will appear a bright halo wheel around the darker object. This is the disadvantage of phase difference microscope, which hinders the observation of fine structures, when the annular diaphragm is narrow, and the halo is more serious. Another phenomenon of phase difference microscope is an asymptotic effect, and when the phase difference observes the same large region, the region edge will decrease in the region.
(3) Effect of Sample Thickness When the phase is observed, the thickness of the sample should be 5 μm or thinner. When using thicker samples, the upper layer of the sample is clear, and the deep layer will be blurred. Unclear and generate phase shift interference and scattering interference of light.
(4) The effect of the cover slide and the slide must cover the cover slide, otherwise the bright ring of the cyclic aperture and the dark ring of the phase plate is difficult to coincide. Partial observation has high quality requirements for glass-slide and cover slides. When there is a scratch, the thickness of thickness or unevenness is unevenly produces bright ring skew and phase interference. The additional slide is too thick or too thin, which makes the annular aperture bright ring or smaller.