Newsgroups: bit.listserv.confocal Date: Wed, 20 Sep 2000 17:21:48 -0400 Sender: Confocal Microscopy List From: Barbara Foster Subject: Re: Koehler illumination To: CONFOCAL@LISTSERV.ACSU.BUFFALO.EDU Vijay, Koehler is the basic alignment procedure. You need to set it each time you sit down at the microscope and touch it up from one area of the sample to another, for optimal results. The protocol varies with each person you talk with. Here's mine" 1. For the eyepieces: (preliminary step) The eyepieces have focus rings on them (turn the very top). Set the "0" to the white dot. 2. For objective: Bring the objective into focus for the sample by "focusing away": Start by looking outside the microscope at the distance between the front of the objective and the sample. Rotate the coarse focus knob away from you to raise the stage as close as possible to the objective. Then, while looking in the microscope, rotate the knob gently toward you until the image comes into sharp focus. Touch up with the fine focus. 3. For the condenser: Look under the stage for the little focusing knob for condenser carrier (usually on the left side of the condenser mount). Raise the condenser until it is just about touching the back of the slide. Note which direction you turned the knob to raise the condenser. a. Close the field iris (the ring around the light port). While watching in the microscope, gently rotate the condenser focus in the other direction until the dark edges of the field iris image come into focus. b. Open the field iris just outside the field of view. (Note: if you have a highly scattering sample, try moving the feature of interest to the center of the field and leaving the field iris mostly closed). c. Adjust the condenser aperture iris (a lever in the condenser if you have a simple condenser or a the little black touch pad on the silver ring around the condenser if you are using a universal condenser) until you have clean, crisp edges and as clean a background as possible. Important note: if you are using a universal condenser (one with many options in a rotating plate), make sure that you set it the "J" to the fiduciary mark on the side. 4. Final setting for eyepieces: a. Do you know which is your dominant eye? If not, email me. Leave the microscope controls set for the dominant eye and adjust the eyepiece focus to bring the image into focus for the other eye. Summary: 1. Set the eyepieces to "O" 2. Focus away with the coarse focus to get a sharp image of the specimen 3. a. Bring the condenser full up to the top using its own focus b. Close the field iris and, while looking in the microscope, focus away to get a sharp image of the field iris superimposed on the image of the specimen c. Set the field iris (usually just outside the field of view) d. Set the aperture iris (sharp, crisp, clean image) 4. Final setting of eyepiece focus for non-dominant eye. This process should take you about 30 seconds. Touch up the two irises as you move from one section of the sample to another or one mag. to another. Because many confocal sections are highly scattering you may only be able to approximate these settings, but doing the best you can will really help the quality of your images and results. Suggestion: see our website for details about our reference book/tutorial, "Optimizing Light Microscopy for Biological and Clinical Laboratories" (www.MME-Microscopy.com/education). Barbara Foster, President Microscopy/Microscopy Education 125 Paridon Street Suite 102 Springfield, MA 01118-2130 PH: 413-746-6931 FX: 413-746-9311 email: MME@map.com Website: www.MME-Microscopy.com/education America's first national consortium of microscopy specialists offering customized on-site training in all areas of microscopy, image analysis, and sample preparation