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					                                                                               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.

PLEASE PRINT NEATLY                      DATE and YEAR Completed/Submitted Test: __________________________
NAME: _________________________________________________________________________________________
STREET: ________________________________________________________________                            APT. _____________
CITY: _________________________________________                   STATE: __________        ZIP: ____________________
PHONE: ____________________________________               Email: _____________________________________________
_____ Yes       _____ No         I am a Michigan Society of Histotechnologists (MSH) member
_____ Yes       _____ No         I have included a check made out to “MSH” in US funds
                                 **FEE for 2 TechPoints:        $5.00 for MSH members, $10.00 for non-MSH members
_____ Yes       _____ No         I require a fee receipt for reimbursement from my employer

Certificate documenting 0.5 hours MSH continuing education (CE) will be mailed to the participant within 4 weeks.

				
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