mikro-graf Volume 39 Fall 2 0 1 0 Issue 04 O ff ic ial Publ ication of the Mich ig an S o c ie t y of Hi stotech nolo g i sts of Histo Seeing The Light with oc iety te ch S Michigan nolo s Kohler Illumination gist Beth Cox, BS, SCT/HTL(ASCP)QIHC Tech Points Ahhhh… think back…remember those brutally hot August days? Remember sitting in the car on the ex- MIcroscoPIc vIew dUrInG Kohler IllUMInATIon pressway in the blazing sun? The sun beating down gave every- thing a strange appearance; the other cars' colors looked washed this issue out and the pavement seemed bent and buckled from the heat waves. The entire horizon looked fuzzy in the glaring light… President’s Message 2 W ell, the explanation is simply too much sunlight; too much uncon- trolled light coming from all directions. The uncontrolled light Editor’s Note 2 washed out the colors and made focusing on objects impossible. The same thing happens when we try to use the microscope without properly controlling its light. Too much uncontrolled, unfocused, random light in the Inbox 3 microscope will bleed out the colors you see and deteriorate the focus, giv- ing you a poor image and making it difficult to evaluate what is present in the specimen. In addition, you’ll probably get a headache from straining your eyes Test Your Knowledge 3 if you try to use the scope that way for very long. Proper alignment of the light in the microscope is achieved by using Kohler Illu- Amyloid Stains 6 mination, originally described by the German scientist, August Kohler, in 1893. Kohler illumination consists of controlling and aligning the beam of light so that it focuses on the specimen, without excessive MSH News & Events 10 light detracting from the image. Using Kohler, an image of the light filament large enough to fill the iris opening is concentrated on the con- Employment Opportunity 11 denser, which is focused so that the iris Meet the MSH Chairs 12 diaphragm image on the lamp is in line Histology Detective 17 with the spec- imen. The lamp iris is AUGUsT Kohler Student Spotlight 20 opened only enough to fill the field of view; the iris of the microscope Region IV & NSH Update 22 is opened only enough to illuminate the back aperture of the objective. In short, while we use the objectives to focus on H&E Slide Analysis 24 the specimen from above, the condenser FocUsInG by condenser & objecTIve should focus the light onto the specimen from below. Officers And Chairpersons 27 [continued on page 4] Mikro-Graf [continued from page 1] before we begin setting up Kohler of illumination. The size of the field Illumination, let's review the parts diaphragm is controlled by rotating a of the brightfield illumination light knurled ring which is on the top of it. microscope involved. some less expensive microscopes have frosted glass lenses in place Condenser of the field diaphragm; these are The condenser concentrates or not controlled by the user and are “condenses” light into a parallel designed to scatter the light rather beam that then passes through than control it. Microscopes without the specimen. It is located imme- controllable field diaphragms should diately beneath the stage of the not be used for critical work or for microscope. The condenser can photography. be raised or lowered to focus the light onto the plane of the speci- Although it would be beneficial to set men by using the knob on the left correct Kohler illumination every time side as you face the scope. do we change the magnification on the not confuse the single condenser microscope, that simply isn’t practi- focusing knob found under the cal. In the real world, Kohler illumina- stage on the left side with the two tion can be set for the most common- sets of coarse and fine focusing ly used objective (usually 10x), and knobs located on both sides of the the swing-in auxiliary lens can adjust lower arm. for use of the 4x objective. This will give a “good enough” setting for daily Two small knurled screws on the front of the condenser are use. since other techs may change the adjustments for used to center it and align the light on the specimen. If the their use, it may be necessary to reset Kohler illumination condenser is not centered, you will see dark and light areas on your laboratory microscope on a regular basis. in your field of view. These will be especially apparent in any photomicrographs you might take. To give the best images for photomicroscopy, it is highly recommended to individually set Kohler illumination for Inside the condenser is an iris diaphragm used to con- each field to be photographed. trol the amount of light that passes through. A lever (or a knurled ring on some microscopes) is used to open and setting up the microscope properly using Kohler illumina- close the leaves of the diaphragm inside the condenser. tion may feel cumbersome the first few times you do it, but This controls the contrast (difference between light & dark) in a short time you’ll be able to quickly adjust and be ready especially at intermediate and high magnifications. The to go. complete instructions are located in the chart (right). condenser diaphragm should not be used to increase or Keep the Quick reference card for Kohler Illumination decrease brightness. If you remove the entire condenser (below) posted near your microscope for easy reference. apparatus (loosen the screws on the sides) and look at it from the bottom, you can open & close the leaves of the Kohler Illumination diaphragm to see how it controls the light. Quick Reference Card Swing-in Auxiliary Lens on the top of the condenser 1. Place a slide on the stage and focus on it sits the swing-in auxiliary lens. (swing out lens should be out for 4x, in for It usually has a knob or lever 10x and 40x). used to swing it in or out of the path of light. This swing-in lens 2. Close both diaphragms. is left out for low-power illumi- 3. Raise or lower the condenser to bring the nation [i.e., 4x], and swung into edges of diaphragm leaves into their sharpest the light path for objectives of 10x or greater magnification. focus. Improper use of this lens is one 4. Center the image. of the most common mistakes that most people make. 5. Open the field diaphragm until the edges just disappear. Field Diaphragm 6. Remove eyepiece and open condeser (upper) The light source is housed in the base of the microscope. The light is redirected by a mirror and passes up through diaphragm until the edges are just inside the the field diaphragm. The field diaphragm controls the area field of view. 4 www.mihisto.org MG39.04 Official Publication of the Michigan Society of Histotechnologists Instructions for Kohler Illumination STEP 1 Place slide on the stage and focus on it. If the microscope is way out of alignment, this may require roughly aligning the illumination by centering the condenser, opening the con- denser aperture diaphragm, opening the field diaphragm, and focusing the condenser to give initially reasonable illumination so you can see well enough to focus on the specimen. (note the dark shadow in the upper right of the picture) STEP 2 close both diaphragms: close the condenser diaphragm using the lever or knurled ring on the condenser. close the field diaphragm located on the base of the microscope to its most closed state. The edges of the image may appear blurry. closing these diaphragms will reduce the illuminated field of view, as seen through the eyepieces, to a small circle of light. STEP 3 Focus the condenser. Turn the focus knob on the condenser so that it moves up or down until the edges of the circle appear sharp. There may be a red fringe on one side of focus and blue on the other: go for the center between these (if the image moves out of your field of view, skip to step 4, then come back to step 3). STEP 4 center the image of the closed field diaphragm in the field of view using the two centering screws on the condenser, so it looks like this. (note centered, crisp edge) STEP 5 open the Field (lower) diaphragm just enough so that its edges just disappear beyond the field of view. (note that the shadow in step 1 is gone.) STEP 6 open the condenser (upper) diaphragm: remove one eyepiece and look into the open tube. you should see an illuminated field of view whose size is controlled by the diaphragm within the condenser. open the condenser diaphragm until its edge is just inside the illu- minated field of view. Then replace the eyepiece. This will adjust the contrast. The amount of contrast added will depend on the sample, however too much contrast can introduce artifacts into your images, too little contrast reduces resolution. References • Albertt einstein college of Medicine, Analytical Imaging Facility, bronx, ny http://www.einstein.yu.edu/aif/instructions/koehler/koehler.htm • dale A. callaham, The central Microscopy Facility, University of Massachusetts, Amherst, MA 01003 UsA • leica Microsystems [online]. richmond hill, ontario. cited september 15, 2010. Available online: http://www.leica-microsystems.com/ • Micrographia; Available online: http://www.micrographia.com/index.htm Fall 2010 www.mihisto.org 5 Earn 0.5 contact hours of continuing education by reading articles in the Michigan Society of Histotechnologists newsletter MIKRO-GRAF. MSH contact hours can be used for CMP required by ASCP BOR to maintain certification. For previous TechPoint articles/tests, go to the MSH website: http://www.mihisto.org Click on Education It is the responsibility of the participant to retain their MSH CE certificates as proof of continuing education. DATE OF ARTICLE: Fall 2010 TITLE: Seeing The Light with Kohler Illumination AUTHOR: Beth Cox, BS, SCT/HTL(ASCP)QIHC DIRECTIONS: 1. Answer the following questions by circling the one (1) BEST answer for each question. 2. Complete the information required at the bottom of the page. 3. Submit questions & check made out to “MSH”(in US funds) to: Peggy Wenk, HTL(ASCP)SLS, 3840 Elmhurst Rd., Waterford, MI 48328 To earn Continuing Education credit from MSH, completed form must be submitted within Three (3) years of original date of the article. 1. To concentrate the most amount of light on the plane of the tissue on the slide, adjust the: A. Field diaphragm B. Fine focus knob C. Objective lens D. Substage condenser 2. The image of the tissue is seen in the upper right of the visual field in the microscope. To center the image, turn the knobs found either on the field diaphragm or on the: A. Eye pieces B. Light source C. Objective lens D. Substage condenser 3. Koehler illumination is required for microscopes using which type of light system? A. Confocal point illumination B. Fluorescence epi illumination C. Phase contrast illumination E. Transmitted bright field illumination 4. TRUE or FALSE (circle one): With Kohler illumination of a light microscope, the image of the light filament is seen on the level of the tissue. 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