Confocal resolution depth

Illumination and contra.st are central points in the discussion of imaging performance. Without sufficient contrast, neither the required magnification nor a maximal resolution can be obtained. A. Kohler developed optical illumination for microscopy in 1893 [8]. The main advantage of Kohler illumination is that in spite of the small filament of the light bulb, the entire area of the lamp field stop, as well as the illuminated part of the specimen, have a uniform luminance (Fig. 4). The twofold ray path of diaphragms and lenses suggests that adjustment of the condenser iris, being the illumination aperture, controls resolution, contrast, and resolution depth. This principle is also important for correct setup of the confocal microscope as well as electron microscopy. 

Depth Depth resolution, depth profiling, slicing, gradients Confocal microscopy, /rATR, SAM... 

The classical polarizing light microscope as developed 150 years ago is still the most versatile, least expensive analytical instrument in the hands of an experienced microscopist. Its limitations in terms of resolving power, depth of field, and contrast have been reduced in the last decade, in which we have witnessed a revolution in its evolution. Video microscopy has increased contrast electronically, and thereby revealed structures never before seen. With computer enhancement, unheard of resolutions are possible. There are daily developments in the X-ray, holographic, acoustic, confocal laser scanning, and scanning tunneling micro-... 

In the z-direction (depth-direction) the resolution is determined by the confocal instrument settings. While it is essential to be aware of the limitations of confocal measurements, as mentioned above, it is possible to create three-dimensional maps. That means probing a sample in the x, y and z-directions. In Figure 2 the data cube of a two-dimensional map is shown. One element (row) of the cube has been picked out and its content enlarged on the right side of the figure. It is evident that each row therefore contains the information of a whole spectrum. 

Two-photon excitation provides intrinsic 3-D resolution in laser scanning fluorescence microscopy. The 3-D sectioning effect is comparable to that of confocal microscopy, but it offers two advantages with respect to the latter because the illumination is concentrated in both time and space, there is no out-of-focus photo-bleaching, and the excitation beam is not attenuated by out-of-focus absorption, which results in increased penetration depth of the excitation light. 

Fig. 2. Depth discrimination (z-axis resolution) properties of a confocal microscope. The illumination and detection images in a confocal microscope are diffraction-limited and confined to a small region of the specimen (1). Only light emitted in the plane of focus and on the optical axis will pass the detector pinhole and form an image. Light emitted from other areas of the specimen does not enter the detector pinhole.

Everall, N. H. (2000) Modeling and measuring the effect of refraction on the depth resolution of confocal Raman microscopy. Appl. Spectrosc. 54, 773-82. 

Figure 3.5-12 Principle of a confocal microscope a LF fiber transporting the laser radiation, D dichroitic mirror, 0 objective, S sample, the Raman radiation produced in the illuminated spot of the sample is focused upon the diaphragm A, only the radiation from the spot is focused at the fiber SF, which transports the Raman radiation to the spectrometer b focal range in the illuminated sample, Ax spatial, Az depth resolution.

While commercial Raman spectrometers collect the scattered light from a laser spot of size 1 pm at best and the Raman spectra also include information of sample regions above and below the focal plane, the implementation of confocal microscopy can improve the optical resolution and the depth of sharpness. The method leads to better image definition. Confocal microscopy means point illumination and that a small aperture is placed in the optical path so that only the scattered light located in a thin focus plane can reach the detector. The aperture-dependent spatial... 

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