1 bring sample into focus with objective focus

1 - bring sample into focus with objective focus knob image point for o 1 and ω2 diaphragm open image point for o 2 and ω1 diaphragm closed 2 - close field diaphragm to minimum size back plane of objective lens front of objective lens 3 - focus condenser until you can see a clear image of the field diaphragm (open and close field diaphragm to check, center as needed) sample (object) condenser lens 4 - open the field diaphragm until the field of view is just completely illuminated condenser diaphragm (focused at back plane of objective) field diaphragm (focused at object plane) collector lens Light source (filament) adapted from Murphy 2001 and Nikon microscopy website http: //www. microscopyu. com/tutorials/java/kohler/index. html Why do we use Kohler illumination? For even intensity at the object and image, we want each small part of our source to contribute to all parts of the image and we want each small part of our image to be made up of light from all areas of the source. We don’t want to see the shape of the light source in our image! What would we see if light rays from A 1 and A 3 were focused at the object/image planes? 5 - now adjust light intensity and the condenser diaphragm to optimize image; small aperture in condenser diaphragm gives best contrast, large aperture gives best resolution (default starting point is for condenser aperture to fill ¾ of objective back plane, this can be viewed with special telescope eyepiece). Notes: Do not adjust light intensity with field or condenser diaphragms, use the condenser diaphragm to optimize contrast and resolution, adjust lamp voltage to correct light intensity.