Review for Basic Microscope Use in the Laboratory

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Review for Basic Microscope Use in the Laboratory

• Graduating Senior Competency Assessment

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Objectives

• Review parts of both compound and dissection
  microscopes.
• Know how to focus a microscope and examine a
  specimen.
• Understand proper microscope care and handling.
• Review basic microscopy concepts and be aware of
  possible solutions and proper protocol when
  problems arise.
• To test your microscope use proficiency before
  graduating. You will be tested through both a
  written as well as a graded skills performance
  test.

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Microscopy Terms

• Field of view lighted circular area seen when
  looking through eyepiece(s).
• Working distance amount of distance between
  objective lens and slide (or specimen).
• Magnification amount of enlargement of image.
• Focus amount of distance between objective lens
  and specimen required for image to appear defined
  and sharp to the human eye.
• Parfocal amount of focus retained when
  switching from one objective lens to anotherthe
  more parfocal the lenses, the less amount of
  focusing needed when objectives are switched.
• Resolution (resolving power) ability to
  distinguish two points of an image as separate.
  Part of clarity of image.
• Depth of field amount of thickness of your
  specimen that is in focus at a single focal
  distance of the microscope.

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Parts of the Microscopes

• In order to use these tools, it is important to
  know what parts comprise each microscope and the
  function of each part.
• Study the next three slides and make sure you can
  identify the basic parts of each microscope.
  Note our Zeiss compound microscopes are
  Fixed-Köhler. This basically means that optimal
  alignment focusing of the light source has been
  pre-set from the manufacturer. Only manipulation
  of amount of light (with diaphragm) or intensity
  (with light switch) is ever needed.

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Parts of a General Compound Microscope
6
Parts of the Zeiss Primostar Compound Microscope
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Parts of the Zeiss Stemi DV4 Dissection Microscope

• Front connection thread M50 to adapt front lens
  systems, ring lamps, and Ø 53mm mount for
  accessories
• Eyepieces 10x20, adjustable for correction of
  ametropiea, if an, individual interpupillary
  distance adjustable
• Eyecups to protect against unwanted ambient
  light, fold over type suitable for spectacle
  wearers
• Carrying handle
• Zoom knob to change magnification
• Focusing knob to enable focusing of the object
• Illumination control panel with three buttons M
  to change the illumination mode and /- to change
  the illumination intensity
• Power-on light, blue
• Integrated reflected-light LED illuminator,
  adjustable
• Clamping screw to fix the Stemi DV4 in the C LED
  stand (SW 3 screwdriver in the stand base
• Integrated transmitted-light LED illuminator
• Glass plate for placing the specimen (also Ø
  84mm insert for stages and transmitted-light/darkf
  ield or contrast/brightfield modules
• Wide-range power supply unit (not illustrated)

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Function of Microscope Parts

• Ocular lens (eyepiece) portal through which
  specimen is viewed also involved in
  magnification of image (ocular magnification is
  stamped on side of eyepiece10X on most scopes).
• Objective lenses involved in amount of
  magnification of image and resolution objective
  magnification is stamped on side of lens (i.e.
  4X, 10X, 40X, 100X) with higher power lenses
  being longer, lower power shorter lenses may be
  dry (uses air as a medium) or oil immersion
  (requires use of oil as a medium between lens and
  coverslip). Oil immersion is used with higher
  magnification lenses (100X)
• Diaphragm regulates amount of light passing
  through specimen.
• Condenser focuses light beam passing through
  specimen.
• Light switch On/off may be used to adjust
  intensity of light.
• Stage where specimen is placed may be
  mechanical to allow precise movement of specimen.
• Coarse fine focus knobs used to move the
  stage (and specimen) vertically to achieve focus
  with a compound microscope.

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The Microscope

• Perhaps one of the most important pieces of
  equipment used by a biologist.
• Treat it with respect and handle it with care.
  They are expensive tools.
• Always carry the microscope with two hands when
  moving it from place to place.
• To prevent internal damage, never scoot a
  microscope across a surface upon which it is
  sitting (rubber feet on bottom are meant to
  prevent this).

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The Microscope

• Compound microscopes use transmitted light and
  require specimens to be mounted on slides.
  Dissection microscopes use either reflected or
  transmitted light, but specimens dont have to be
  mounted on slides.
• For compound microscopes, specimens are placed on
  a glass slide with a cover slip in place.
• After use of an oil immersion objective lens,
  NEVER return a dry lens into position to use
  without first cleaning all of the oil off of the
  slide and oil immersion objective. Failure to do
  so contaminates the dry lenses with oil renders
  them unusable, possibly permanently.

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The Microscope

• All microscopes are not made exactly the same,
  but they should have the same basic components as
  you saw in the previous microscope figures. You
  should be able to recognize the basic parts on
  different models.
• You should be able to apply your knowledge about
  use of a compound microscope to properly use a
  dissection microscope. Key differences are
  pointed out in this presentation.
• If you have learned how to use our microscopes,
  you should be able to use any others in future
  situations or jobs. If you have problems with use
  of a particular microscope in the future, consult
  the operators manual.

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Where to Begin

• Carefully remove microscope from storage cabinet
  and carry with both hands to your table. Make
  sure not to bump it into any surfaces upon
  removal or while transporting to table.
• Microscope should be gently placed onto table
  surface in order to prevent any kind of jarring
  impact that may misalign scope optics and render
  it unusable.
• Remove dust cover. Make sure microscope is placed
  on table in a position for you to be able to view
  through eyepiece(s). If it needs to be moved, DO
  NOT scoot it across the table surface! Pick it up
  and gently reposition it.

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Where to Begin (continued)

• Plug in the microscope, turn on the light switch,
  and adjust the light intensity. Close/open
  diaphragm about half way to start.
• Before placing slide on stage, make sure the low
  power objective is securely clicked into place.
• Use coarse focus knob to create enough distance
  between objective lens and stage to safely load
  the slide onto the stage. Make sure coverslip on
  slide is facing upward. Dont look through the
  eyepieces when adjusting the coarse focus knob.
  Look at the stage.

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Where to Begin (continued)

• Place slide on stage making sure to properly use
  slide holders to hold slide in place. Do not let
  spring-loaded holders flip back quickly against
  edge of slide or slide may break.
• The two knobs of the mechanical stage should then
  be used to position your specimen over the
  opening in the stage where light is coming
  through.
• Now adjust coarse focus knob (again viewing from
  the side) to where objective lens is close to
  slide, but not touching it. If adjusted too fast,
  the slide could make contact with the objective
  lens and break the lens or slide.

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Where to Begin (continued)

• Make sure you are aware of what direction the
  stage moves when you move either of the focus
  knobs. (i.e. when you turn the knob toward you,
  is there moreor is there less distance between
  objective and slide?). You need to be aware of
  this in order to prevent possible damages while
  operating the microscope.
• Adjust interocular distance between eyepieces so
  that you are comfortably viewing a perfect
  circular field of view. Do not try to view
  specimen through only one of the eyepieces! If
  you feel you need to do that in order to view
  the specimen, you dont have the interocular
  distance properly adjusted for your eyes.
• Looking through eyepieces, focus on specimen
  first with a small amount of adjustment with
  coarse focus, followed by focusing with fine
  focus to get best focused image.

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Where to Begin (continued)

• You may need to adjust focus of individual ocular
  lenses if one eye seems out of focus to the
  other. Some eyepieces have focusing rings
  encircling them, which can get out of adjustment
  and make one eye unfocused compared to the other.
  
• To correct this, first close one eye and focus
  the other eye by using the focus knobs on the
  body of the scope to bring the open eye into
  focus.
• Then close the eye on the focused side and open
  the opposite eye. If the opened eye is unfocused,
  twist the focus ring on that eyepiece until a
  crisply focused image is achieved. Now open both
  eyes and you should see a focused image with both
  eyes opened at the same time.
• If you have done this properly, there should be
  no need to use the eyepiece focusing rings after
  this point during this particular viewing session
  (as long as no one else is using this microscope
  and has changed your setting).
• NOW, you should be ready to start using the
  microscope.

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Using the Microscope

• Scan the specimen (notice direction each
  mechanical stage knob moves stage and compare to
  viewed specimenit will be opposite direction)
  using lowest power objective first. Focus with
  fine focus and adjust light with diaphragm as
  needed.
• Realize you can focus on any individual plane
  from top through bottom of your specimen, but
  only one plane at a time. Make sure you can
  determine depth orientation of a focused part of
  a specimen in comparison to other parts (i.e. is
  a focused part above or below another part in the
  specimen).
• Find an area of interest that you want magnified
  greater. Move the area of interest of your
  specimen directly into the center of your field
  of view and switch to next higher power objective
  and focus with fine focus knob for a closer look.
  Use only fine focus knob with higher power
  objectives in place.
• Realize more light is needed as you increase
  magnification, so adjust diaphragm accordingly.

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Using the Microscope (continued)

• If you lose your specimen in the field of view,
  always go back to the next lower power objective
  to relocate your specimen or area of interest and
  make sure it is centered in the field of view
  before trying higher power again.
• If you still need greater magnification of an
  area of interest, center that area and rotate the
  next higher power objective into position (as
  long as it is a dry lens and not oil immersion)
  and focus with fine focus and adjust amount of
  light.
• If the next higher power objective is an oil lens
  (look for stamp of oil on side of objective),
  immersion oil is required to be added on top of
  the cover slip before the oil lens is clicked
  into position to create a bridge of oil between
  the lens and cover slip. Always remember, NEVER
  go from using an oil lens back to a dry lens,
  without first thoroughly cleaning the oil from
  the slide and oil lens!

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How to Finish

• After you have finished examining a specimen, the
  lowest power objective lens must be clicked into
  position.
• Make sure there is enough distance between slide
  and objective lens to prevent accidental contact
  while unloading slide from slide holders on
  stage.
• Wipe the stage area clean if any liquid or solid
  materials remain after removing the slide that
  was viewed.

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How to Finish (continued)

• Make sure lenses are clean (see care of
  microscope).
• Turn off light switch, THEN unplug electrical
  cord.
• Make sure cord is properly stored on microscope.
• Place microscope dust cover over microscope.
• Return to proper storage area by carefully
  carrying it with both hands and making sure not
  to bump it into anything.
• Microscope should be gently placed onto cabinet
  surface where it is stored in order to prevent
  any kind of jarring impact that may misalign
  scope optics and render it unusable.

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Cleaning and Care of the Microscope

• Always make sure the eyepieces, objectives, and
  light source are clean before, during, and after
  use.
• Only use Lens Paper to clean these glass surfaces
  to prevent scratching.
• The lenses or eyepieces may easily become dirty
  especially if many different people use the same
  microscope. Eye make-up may build up on the
  lenses or glasses may scratch them. Clean them
  often.

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Cleaning and Care of the Microscope (continued)

• If there is no light from light source after
  turning on light switch and increasing light
  intensity, the bulb is probably burned out and
  needs to be replaced.
• When the microscope is not in use, it is always
  good practice to place the dust cover over the
  instrument to prevent contamination on lenses and
  other surfaces.
• Without daily maintenance a microscope can be
  very costly to repair.

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Microscopy Concepts and Practices You Should Know

• Total magnification of a specimen is calculated
  by multiplying ocular lens magnification by the
  magnification of the objective lens clicked into
  position. This informs you how much larger the
  image you are viewing through the eyepieces is in
  comparison to the actual size of the specimen
  being viewed.
• When you increase magnification
• - field of view decreases
• - working distance decreases
• - amount of light needs to be increased
• - depth of field becomes shallower

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Microscopy Concepts and Practices You Should Know
(continued)

• Image in a compound microscope is inverted (i.e.
  upside down and backwards) compared to actual
  specimen on slide. This is due to use of a single
  objective for both eyes with this type of
  microscope. Image is 2-dimensional.
• Image in a dissection microscope is in the same
  orientation as the actual specimen. This is due
  to use of separate objective lenses for each eye
  to produce a separate image for each. Viewed
  Image is therefore stereoscopic and
  3-dimensional. This type of microscope has only
  one focus knob (i.e. lacks a separate fine focus
  knob)

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Microscopy Concepts and Practices You Should Know
(continued)

• Transmitted light is used to illuminate a
  specimen by passing through the specimen. This is
  why clearing and/or staining of the specimen may
  be needed to view, or see details. This type of
  lighting is used with compound, as well as some
  dissection microscopes.
• Reflected light illuminates a specimen by
  reflecting off its surface. Angle and intensity
  of light is important. This type of lighting is
  used with dissection microscopes.

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Microscopy Concepts and Practices You Should Know
(continued)

• Always start viewing with the lowest power object
  lens in position because
• - gives greatest field of view making it easier
  to initially scan slide and find specimen
• - has greatest working distance making it
  easier
• to load slide without damaging it
  or objective
• Never use coarse focus knob when a high power
  objective lens is in position because the coarse
  knob causes larger amounts of movement when there
  is an extremely small working distance. This may
  result in contact between the slide and
  objective, possibly producing damage.

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Microscopy Concepts and Practices You Should Know
(continued)

• Always directly center a specimen (or area of
  interest in a specimen) in the middle of the
  circular field of view before changing to the
  next higher power objective lens. This is done
  each time you change to a higher power objective.
  This will help prevent loss of specimen at
  higher magnification.

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Problems What Should I Do When?

• I turn on the light switch and there is no light?
• POSSIBLE ANSWER
• 1.)Light intensity may be set too lowincrease
  light intensity.
• 2.)Bulb may be burned outreplace bulb.
• 3.)Poor electrical contact of bulb or short in
  electrical cordrepair required.
• One eye is in focus and the other is not?
• POSSIBLE ANSWER
• Ocular focus not properly adjusted for out of
  focus eye of usertwist ocular focus ring on
  eyepiece to achieve focus.

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Problems What Should I Do When? (continued)

• I dont see a field of view that forms a complete
  circle when looking through both eyepieces at the
  same time?
• POSSIBLE ANSWER
• Interocular distance between eyepieces is not
  properly adjusted for distance between eyes of
  usermove eyepieces in or out until a perfect
  circular field of view is achieved.

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Problems What Should I Do When? (continued)

• I see either a portion of the field of view
  darkened or no field of view (even though light
  is obviously turned on and shining)?
• POSSIBLE ANSWER
• 1.)Objective lens is not properly
  positionedmake sure lens is clicked into
  position.
• 2.)Condenser is misaligned and improperly
  focused (if not fixed Köhler)align and focus
  condenser.
• 3.)Electrical cord is lying across light
  sourcerelocate path of cord.

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Problems What Should I Do When? (continued)

• I cant find my specimen (or area of interest of
  specimen) after changing to a higher power
  objective lens to get greater magnification?
• POSSIBLE ANSWER
• 1.)Specimen was not centered in field of view
  before switching objectivesgo back to next lower
  power objective and properly center specimen
  directly in middle of field of view.
• 2.)Lens may be dirty or damagedclean lens or
  replace damaged lens.

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Problems What Should I Do When? (continued)

• The image seen with the high dry objective lens
  is extremely distorted (similar to looking at
  something underwater)?
• POSSIBLE ANSWER
• 1.)Lens has been contaminated with immersion oil
  from improper use of microscope by another
  userremove and carefully and thoroughly clean
  lens. It is imperative that you do not allow
  anything to enter open end of objective.
• 2.)Lens is otherwise dirty or has debris
  adhering to itclean lens thoroughly.

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Final Thoughts

• Now that you have completed this auto-tutorial
  slide presentation, you will have part of a class
  meeting set aside to actually practice with the
  microscopes in lab.
• The following week is when we will have the
  graded microscope use proficiency test that is
  comprised of both written and actual skills
  performance components.
• This graded test will count as a portion of your
  final grade for BIOL 4001 Seminar in Biology.
• If you have any questions about this material or
  use of the microscope, please ask your instructor.