Holographic interference microscopy (HIM) is holographic interferometry applied for microscopy for visualization of phase micro-objects. Phase micro-objects are invisible because they do not change intensity of light, they insert only invisible phase shifts. The holographic interference microscopy distinguishes itself from other microscopy methods by using a hologram and the interference for converting invisible phase shifts into intensity changes. Other microscopy methods related to holographic interference microscopy are phase contrast microscopy and holographic interferometry.
Holographic interference microscopy methods Holography was born as "new microscopy principle". D. Gabor invented holography for electron microscopy. For some reasons his idea is not applied in this branch of microscopy. But invention of holography opened up new possibilities in imaging of phase micro-objects due to the application of the holographic interference methods in microscopy that allow not only qualitative, but quantitative study. Combining the holographic interference microscopy with methods of numerical processing has solved the problem of 3D imaging of untreated, native biological phase micro-object. In the holographic interference method the images appear as the result of the interference of two object waves passed the same path through the microscope optical system but in different points of time: the reconstructed from the hologram "empty" object wave, and the object wave disturbed by the phase micro-objects under study. The hologram of the "empty" object wave is recorded using a reference beam, and it is used as an optical element of the holographic interference microscope. In the dependence on conditions of the interference two methods of the holographic interference microscopy can be realized: the holographic phase-contrast method and the holographic interference-contrast method. In the first case, the phase shifts inserted by the phase micro-object into the light wave passing through it are converted into intensity changes in its image; and in the second case – into deviations of interference fringes.
Holographic phase-contrast method Holographic phase-contrast method is the holographic interference microscopy technique for phase micro-object visualization that converts the phase shifts inserted by the micro-object to the wave of light passed through it into intensity changes in the image. The method is based on the holographic addition (constructive interference) or holographic subtraction (destructive interference) of the "empty" wave reconstructed from the hologram, and the object wave disturbed by the phase micro-objects under study. The image can be considered as interferogram in interference fringes of infinite width.
The method solves the same problem as does F. Zernike phase contrast method. But in comparison with F. Zernike phase contrast method, the method has some advantages. Due to equal intensities of the interfering waves, the holographic phase-contrast method allows obtaining maximal contrast of images. The sizes of the micro-object do not restrict the application of the method, though F. Zernike phase contrast method works the more successfully, the smaller the micro-object in thick and sizes. The image in the holographic phase-contrast method is the result of interaction of two identical waves, and it is free of aberrations. The method can be realized as the method of holographic addition and subtraction in an interference fringe. A small angle is introduced between the interfering waves so that the period of resulting system of interference fringes significantly exceeds the size of the images. The conditions for the waves to be antiphased or in-phased (holographic subtraction or addition) are automatically created within a dark and bright interference fringes, correspondingly. The intensities in the image of the micro-object I i m + {\displaystyle I_{im+}} and the intensity of the background I b + {\displaystyle I_{b+}} in the case of wave addition in a bright interference fringe are determined by the expressions:
I i m + ( x ′ , y ′ ) = 2 I 0 [ 1 + c o s f ( x , y ) ] {\displaystyle I_{im+}(x',y')=2I_{0}[1+cosf(x,y)]} ; I b + = 4 I 0 {\displaystyle I_{b+}=4I_{0}}
and the intensities in the image of the micro-object I i m − {\displaystyle I_{im-}} and the intensity of the background I b − {\displaystyle I_{b-}} in the case of wave subtraction in the dark interference fringe (waves are antiphased):
I i m − ( x ′ , y ′ ) = 2 I 0 [ 1 − c o s f ( x , y ) ] {\displaystyle I_{im-}(x',y')=2I_{0}[1-cosf(x,y)]} ; I b − = 0 {\displaystyle I_{b-}=0}
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