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Images: Process Microscope

Magnification, resolution, distortion, contrast

Figure 1 is a section taken from the image of a fine metric scale, submersed in water. This image was obtained with the ULTROPAK 11 objective. The lines of the scale have a spacing of 10 µm. They are well resolved, indicating that the microscope has a resolution Dx well below 10 µm. The width of the lines is estimated to be 2...3 µm. A resolution of this kind must be expected, confirming the high-quality of the microscope objective. From the complete image of this metric scale the dimensions of the field of view have been determined. They were found to agree with the size calculated for this objective (1.33 x 1.00 mm²). Figure 2 shows individual bubbles of air in water. Their sizes vary from 100...300 µm. This example indicates some of the basic difficulties in imaging transparent objects. Because the bubbles have a perfectly smooth surface, their front surfaces act like tiny convex mirrors, and likewise their rear surfaces are concave mirrors. According to the difference of the refractive indices of air and water, the reflectivity of these 'mirrors' is only 2%. Nevertheless, this image Figure 2 is well exposed, thus confirming the estimated power budget given above. The most prominent features of Figure 2 are the dotted rings. Each bubble shows 2 concentric rings with 18 discrete spots. They are easily understood to result from the mirror action of the bubbles. The spots are demagnified images of the 18 fiber ends which carry the illumination light. The regular appearance of these dots indicates the correct alignment and operation of the illumination system. If one of the fibers were broken, the corresponding spot on the bubble would be missing. In this sense the bubbles are recognised as a valuable tool for checking the microscope system.

	Figure 1. Section cut from the image of a millimeter scale, obtained with the 'cold' microscope, using the 11x lens. The spacing of the lines is 10µm. The lines are well resolved, indicating that the resolution in this microscope image is at least of the order of 2 µm.
	Figure 2. Image of air bubbles in water, obtained with the 'cold' immersion microscope, using the 11x lens. The bubbles act as perfect little convex mirrors, reflecting the illuminating light. It can be seen that this light comes from 18 distinct sources (from the 18 fibers).

Illumination intensity, depth of field, scattering objects

Figure 3 is the image of a thin metal mesh of square symmetry and periodicity 100 µm. This type of image serves to analyse distortions which may possibly have been introduced by the imaging system. Obviously, as the lines of the mesh appear perfectly straight, any distortion present is very small, not recognisable by the naked eye. Besides the absence of distortions, Figure 3 shows that the distribution of the illuminating light is nonuniform across the field of view. A central region of the field, measuring approximately 600 µm in diameter, receives much more light than regions near the edges. This effect increases the detectability of small scattering particles in the center, and this bright central spot has actually been used in setting up the power budget. The following Figure 4 and Figure 5 are obtained with small, transparent crystals (ascorbic acid, C₆H₈O₆) dispersed in water and suspended by rapid stirring. The resulting motion of the particles has a velocity of ~1 m/s, as found in the biogas reactor. Despite this motion, the images show some particles well focused, indicating that the concept of 'freezing' the motion by flash illumination does work. Next to those particles, however, also a number of blurred particles can be seen. They are interpreted as being out of focus, because the probe volume has a considerably wider extent than the depth of field. Nevertheless, the number of blurred particles is not vastly larger than the number of well-focused particles, indicating that the mentioned difference in the depths is only moderate. Figure 6 is an image obtained in air. Here, clearly, the concept of dark field illumination fails. Rather, an external tungsten lamp has been the light source in taking this image.

	Figure 3. Image of a metal mesh structure with a periodicity of 100 µm. This picture shows that there is essen-tially no image distortion, and that the illumination is not uniform. By the focussing action of the axicon lens, the available illumination energy is concentrated into a central region of 600 µm diameter. This improves the imaging of weakly scattering particles, e.g. cells in water.
	Figure 4. Image of colourless transparent crystals (ascorbic acid, C₆H₈O₆ ) suspended in water by stirring. The smallest crystals have a size of ~ 30 µm. This picture illustrates the imaging of moving objects (velocity v ~ 1 m/s). The motion is 'frozen' by the flash illumination (t ~ 3 µs). The resulting motion blur is ~ 3 µm and hardly discernible in this picture.
	Figure 5. Another picture of a crystal of ascorbic acid in water. This view gives an imression of the 3rd dimension: The crystal to the left happens to be 'in focus', another object to the right (another crystal ?) is in the background and appears diffuse due to 'defocusing'.
	Figure 6. Example of a still 'dry' image, obtained with the microscope in air: The surface of white paper, with part of a letter printed by a laser printer. The fibrous structure of the paper and individual grains of the black 'toner' can be distinguished. The printed line has a width of ~ 170 µm.

Performance with biological objects, fluorescent imaging, artefacts

Finally, the Figure 7 and Figure 8 show two images of a suspension of algae of the kind Pseudo Kirchneriella Subcapitata. This sample has been selected because it is known to emit red fluorescent light when excited in the blue. The first image is relatively blurred because the cell concentration is fairly high here, and the fluorescent light cannot be seen. Rather, when viewed on a colour monitor, Figure 7 appears intensely blue-green, because the illuminating light has been filtered here with a short-pass filter, blocking all radiation with wavelengths longer than 455 nm. However, when observed through a red filter, the fluorescence becomes clearly visible, because the red filter blocks all direct illuminating light. Figure 8 shows many fine spots, glowing intensely red, representing the fluorescent light. The last image, Figure 9, has been included here because it shows a familiar object with a strange structure. The grains of Corundum (on abrasive paper) all appear doughnut-like, with a hole in their center. We believe this appearance to result from the dark field illumination. In that arrangement, no light is incident normally onto the sample. Therefore, the tops of the generally convex grains remain dark, comparable to the 'tops' of the air bubbles in Figure 2.

	Figure 7. Image of nearly transparent algae (pseudo Kirchneriella subcapitata) suspended in water, illuminated by the flash lamp through a blue filter, (lc ~ 455 nm), observed without filtering. The smallest discernible cells are ~ 2...3 µm in size. This picture illustrates the possibilty of regognizing cells selectively by their fluores-cence. (see Figure below)
	Figure 8. Illustration of 'fluorescence imaging': The same scene as above, illuminated through a blue filter (blocking red light), and observed through a red filter (blocking the blue illuminating light). The fine dots visible in this image appear intensely red. They represent red fluores-cent light, generated in the cells under excitation by the blue illuminating light. The fluorescent cells are estimated to be <= 2 µm in size.
	Figure 9. Grains of corundum, Al₂O₃ (surface of abrasive paper), with an average particle size of ~ 100 µm. The dough-nut-like appearance of the particles is an artifact, resulting from the dark-field illumination.