Reflected Light Microscopy

In reflected light microscopy, the specimen is illuminated and imaged from the same side. This technique is commonly used for metallic specimens. When placing the polished specimen in the microscope the surface to be examined optically should be perfectly flat and level. If not, then as the viewing area is moved across the surface it will pass in and out of focus and it will make it difficult to have the whole of the field of view in focus. Provided the polished surface is planar, this problem can be avoided using a specimen levelling press, Figure 12. The specimen is placed on a blob of plasticene on a microscope slide, and pressed to make it level. A small piece of paper or cloth covers the surface of the specimen to avoid scratching.

Figure 12 – A specimen levelling press

Once the specimen had been mounted on the slide, it can be imaged. A typical reflected light microscope is shown in Figure 13, and standard protocol for using any optical microscope is given below:

1) Place the mounted specimen on the stage. The area to be viewed should be positioned approximately central on the stage, beneath the objective lens.

2) If the microscope is binocular (has two eyepieces) adjust the eyepieces so they are the right distance apart.

3) Switch on the illumination, starting at a low power setting and gradually increase until the desired light level is reached. This can best be judged by increasing the power until a clear spot of light is seen on the specimen surface.

4) Use the lowest magnification objective lens to coarsely focus the microscope. To avoid “crashing” the lens into the surface of the specimen start by bringing the lens close to the surface looking from the side. Then, looking down the microscope, focus by pulling the lens away from the sample surface.

5) Looking through the eyepieces, use the fine focus knob to bring the image into sharp focus.

6) On a binocular microscope, the eyepieces can now be further adjusted; one of them will focus separately, as many people have slightly different focus in each eye. The image should be brought into focus in the non-adjustable eyepiece first using the fine focus knob, and then the other eyepiece adjusted so that it is also in focus.

7) If a higher magnification is required the appropriate lens can be moved into place, and the image can be refocused using the fine focus knob. When using higher magnification lenses, the coarse knob should not be used for focussing, due to the risk of crashing the lens in to the specimen.

8) If different areas need to be imaged, many microscopes have mechanical controls that allow repositioning of the specimen under the lens. If the specimen has not been mounted flat, making such movements at high magnification brings the risk of crashing the lens into the sample.

Figure 13 – A reflected light microscope

Дата добавления: 2015-10-26