Contrast Enhancement with the Light Microscope

1. Many biological structures are very difficult to see under the microscope. Some can be stained with specific dyes or labeled with fluorescent tags but others simply require optical contrast enhancement strategies. There are a variety of enhancement techniques that can be used with the light microscope including phase contrast, Nomarski differential interference contrast, Rheinberg illumination and darkfield illumination.
   
2. The simplest, but definitely not the best, way to gain contrast involves reducing the diameter of the microscope's aperture diaphragm beyond optimum in order to increase contrast at the expense of resolution. This also increases the depth of field but, because of diffraction limits, the cost on resolution is almost immediately apparent.
   
3. Köhler illumination is the normal way of setting the controls of the microscope for bright field illumination. First, the condenser top should be removed and the condenser itself should be lowered when you are using objectives less that 10x magnification. For all other objectives the condenser top should remain on and the whole condenser should be near the upper limit of its range.
   
4. Both the field diaphragm (in the base of the scope) and the aperture diaphragm (in the condenser) should be opened and the rotating condenser turret should show J opposite its right side index line.
   
5. Search your slide using a lower magnification objective and increase magnification as necessary. If you use the 100x lens use immersion oil to join the lens to the slide. No other lens may at any time come in contact with oil. To oil the lens put a tiny drop on the lens and a small drop on the slide. Then rotate the lens so that the two drops merge.
   
6. When you have found the wanted field of view, focus the image of your material, close the field diaphragm until it is visible through the binocular eyepieces, focus its image with the condenser drive and center it using the two large adjustment screws on the condenser carrier. Open the diaphragm again until it just vanishes from view.
   
        
   
7. Next, for brightfield observation, remove one eyepiece and close the aperture diaphragm (in the condenser) until it darkens or occludes the outer quarter of the bright circle which is visible down the tube. These steps establish Köhler illumination, first described by Dr. August Köhler over 100 years ago.
   
8. In order to use phase contrast both the objectives and the condenser must be phase capable. The objectives will have small dark phase retarding rings and the condenser will have several positions with light transmitting rings of diameters that optically match that of the corresponding objective.
   
9. To set up for phase, first rotate the condenser turret so that 2 is opposite the index mark on the right side of the condenser when using the 16x Ph and 40x Ph objectives or 3 is opposite the index when using the 100x Ph objective. Replace one eyepiece with the phase telescope (which is focused by sliding the top half of it in and out in relation to the other half). The positive phase ring should now be superimposed on the negative ring by using the two adjustments on the condenser, the one nearest you for east-west movement and the one on the left for north-south movement. Material that causes a significant phase shift will result in strong halo like artifacts that can obstruct parts of the image. In those cases Nomarski may be a better technique.
   
        
   
10. Nomarski Differential Interference Contrast (DIC) involves different strategies that require even more specialized optics than phase contrast. Changes in the refractive index of the specimen are made visible as three dimensional relief. The effect of the relief will vary with rotation, so ideally the scope should have a rotating stage that allows rotation of the prep.
    
11. A microscope equipped for Nomarski will have a polarizer and a modified Wollaston prism below the condenser and a similar pair will be located above the specimen, with the second polarizer above the second Wollaston. A polarizer is a filter that restricts the vibration of light into a specific plane. A Wollaston prism consists of two calcite prisms arranged so that they deviate two emerging beams of light that are polarized at right angles to each other in opposite directions. As a result of the pairing of Wollastons below and above the specimen, light is sheared, passed through the prep and the wavefront is then recombined, with interference strengthening the image. The top prism is adjustable, with a small screw to move it across the light path, and the amount of DIC can be changed by moving it back and forth.
    
12. First Köhler Illumination should be established. Then the substage polarizer should be set east west and the condenser setting selected, depending upon which objective is to be used. Position I is for the 16x plan objective, position II is for a 40x plan lens and III is for the 100x plan lens. The Nomarski analyzer should be in the light path above and should be moved across the light path, until the best image results.
    
    

    
    
    
    
13. Rheinberg filters are one of the simplest things you can make and colour pictures made with them can be quite stunning. The filters can be made of thin coloured acetate and might look like these examples. The large filter's outer diameter should be that of your substage filter holder and it will need a hole cut in it of about 17mm diameter. A disk of a complementary colour should be fitted into the hole so that the result might look like:
    
    
14. When a refractile specimen is viewed with the Rheinberg filters in place it will be seen against the central colour while its outer surfaces will be fringed with the outer colour.
    
15. Another technique that works well for transparent materials is darkfield illumination. Some rotating condensers have a darkfield stop, otherwise a darkfield condenser can be used in place of a standard condenser. The result will be a dark background against which the specimen will appear light and jewel like.
    
16. In addition to the microscope based contrast enhancement tools and techniques mention there are many darkroom or computer based post processing strategies.
    
    An original micrograph is shown in figure a. In figure b, the active layer has been duplicated in Photoshop, processed with the Reindeer Graphics Adaptive Equalize filter and the two layers were then blended via the soft light mode. In figure c the original image was treated with a levels adjustment in which the black and white points were brought up to the limits of the histogram as seen below. In figure d the original image layer (layer 2) was duplicated (layer 2 copy) and the layers were then blended with the multiply mode.
    
    
    
    There are many other ways of improving images in the computer, often by copying the image, modifying the copy by applying an algorithm to it and then blending the copy with the original.