Introduction to Microscopy

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Introduction to Microscopy

• The basics and beyond

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What is a microscope? How does it work?

• A microscope is an instrument designed to make
  fine details visible.
• The microscope must accomplish three tasks
• produce a magnified image of the specimen
  (magnification),
• separate the details in the image (resolution),
  and
• render the details visible to the eye, camera, or
  other imaging device (contrast).

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The Human Eye

• Our eyes are capable of distinguishing color in
  the visible portion of the spectrum from violet
  to blue to green to yellow to orange to red the
  eye cannot perceive ultra-violet or infra-red
  rays.
• The eye also is able to sense differences in
  brightness or intensity ranging from black to
  white and all the grays in-between.

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Light

• What is light and how does it interact with
  matter?
• Light has both particle and wave natures.
• For microscopists it is the wave theory that is
  applied in most cases.
• Wavelength of light is perceived as colour and
  the amplitude is seen as brightness.

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Light

• Light travels in straight lines
• Its path can be deflected or reflected by means
  of mirrors or right angle prisms.
• Light can be bent or refracted by means of
  glass lenses that are thicker or thinner at their
  center or their periphery.
• Light travels at different speeds in air and in
  glass (faster in air which is usually taken as
  the standard of 1).
• Light is slowed and bent or refracted when it
  passes through air and enters a lens.

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Lenses

• The two most common types of lenses are concave
  and convex. A common bi-convex lens is considered
  a positive lens because it causes light rays to
  converge or concentrate forming a real image.

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Focal point and image formation

• Every convex lens has two focal points one on
  each side of the lens. If an object is placed on
  the far side of the front focal point (F), an
  inverted image is projected on a screen placed on
  the opposite side of the lens. The inverted image
  is known as the real image.
• If the object is brought to a point between the
  focal point and the lens, it appears enlarged and
  is transposed to the same side of lens as the
  object. The enlarged image is known as the
  virtual image.

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Focal point and image formation
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SIMPLE MICROSCOPE

• More than five hundred years ago, simple glass
  magnifiers were developed.
• These were convex lenses (thicker in the center
  than the periphery). The specimen or object could
  be focused by use of the magnifier placed between
  the object and the eye.
• These simple microscopes, along with the cornea
  and eye lens, could spread the image on the
  retina by magnification through increasing the
  visual angle on the retina.

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SIMPLE MICROSCOPE
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Compound Microscope

• In the compound microscope we find the object
  that is magnified by the object (forming the real
  image) is further magnified by the ocular lens to
  form the virtual image.
• When you look into a microscope, you are not
  looking at the specimen, you are looking at an
  IMAGE of the specimen.
• The image is floating in space about 10 mm
  below the top of the observation tube (at the
  level of the fixed diaphragm of the eyepiece)
  where the eyepiece is inserted.

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COMPOUND MICROSCOPE

• The compound microscope achieves a two-stage
  magnification. The objective projects a magnified
  image into the body tube of the microscope and
  the eyepiece further magnifies the image
  projected by the objective (more of how this is
  done later).
• For example, the total visual magnification using
  a 10X objective and a 15X eyepiece is 150X.

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Types of Microscopes
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Optical Components

• The most important optical component of the
  microscope is the OBJECTIVE.
• 1. Its basic function is to gather the light
  passing through the specimen and then to project
  an accurate, real, inverted IMAGE of the specimen
  up into the body of the microscope.
• 2. Other related functions of the objective are
  to house special devices such as an iris for
  darkfield microscopy, a correction collar for
  counteracting spherical aberration or a phase
  plate for phase contrast microscopy.
• 3. The higher power objectives should have a
  retractable front lens housing to protect the
  front lens where the objective requires focusing
  very close to the specimen.
• 4. To the extent possible, corrections for lens
  errors (aberrations) should be made within the
  objective itself.

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Examples of Objective Lens
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Optical Components

• A second important optical component is the
  EYEPIECE or Ocular Lens.
• 1. Its basic function is to look at the
  focused, magnified real image projected by the
  objective and magnify that image a second time as
  a virtual image seen as if 10 inches from the
  eye.
• 2. The eyepiece can be fitted with scales or
  markers or pointers or crosshairs that will be in
  simultaneous focus with the focused image.

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Optical Components

• The third important optical component is the
  SUBSTAGE CONDENSER.
• 1. Its basic function is to gather the light
  coming from the light source and to concentrate
  that light in a collection of parallel beams onto
  the specimen.
• 2. The light gathered by the condenser comes to a
  focus at the back focal plane of the objective.
• 3. In appropriately set up illumination the image
  of the light source comes to focus at the level
  of the front focal plane of the condenser.
• 4. Correction for lens errors are incorporated in
  the finest condensers, an important feature for
  research and photography.
• 5. Where desired, the condenser can be designed
  to house special accessories for phase contrast
  or differential interference or darkfield
  microscopy

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Examples of Condensers
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Mechanical/Electrical Components

• The STAND of the microscope houses the
  mechanical/electrical parts of the microscope. It
  provides a sturdy, vibration-resistant base for
  the various attachments.
• The BASE of the microscope is Y-shaped or
  U-shaped for greater stability. It houses the
  electrical components for operating and
  controlling the intensity of the lamp. The lamp
  may be placed, depending on the instrument, at
  the lower rear of the stand or directly under the
  condenser fitting. The base also houses the
  variable field diaphragm.

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Mechanical/Electrical Components

• Built into the stand is a fitting to receive the
  microscope STAGE. The stage has an opening for
  passing the light. The specimen is placed on top
  of the stage and held in place by a specimen
  holder. Attached to the stage are concentric X-Y
  control knobs which move the specimen forward
  /back or left/right.
• On the lower right and left sides of the stand
  are the concentric COARSE and FINE FOCUSING
  KNOBS. These raise or lower the stage in larger /
  smaller increments to bring the specimen into
  focus.

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Mechanical/Electrical Components

• 4. Under the stage there is a built-in ring or a
  U-shaped CONDENSER HOLDER. Adjacent to the
  condenser holder there are either one or two
  knobs for raising or lowering the condenser.
• 5. Above the stage, the stand has a NOSEPIECE
  (may be fixed or removable) for holding the
  objectives of various magnifications. The
  rotation of the nosepiece can bring any one of
  the attached objectives into the light path
  (optical axis).

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Using the microscope
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A method for setting up the microscope correctly

• Köhler illumination was first introduced in 1893
  by August Köhler of the Carl Zeiss corporation as
  a method of providing the optimum specimen
  illumination.

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Setting Up Köhler illumination

• After switching on the lamp of the microscope,
  open up fully both the field diaphragm (in the
  light port of the microscope) and the
  iris/aperture diaphragm (usually built into the
  substage condenser). Check you have light by
  insert a piece of paper over the light.

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Setting Up Köhler illumination

• Then set the interpupillary distance via the
  folding bridge of the binocular tube. The correct
  setting is reached when you see one light circle
  instead of two.
• Rotate the nosepiece to bring the 10X objective
  into the light path.
• Place the specimen on the microscope stage and
  focus the specimen using the coarse and fine
  focusing knobs.

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Setting Up Köhler illumination

• Close down the field diaphragm most of the way.
  Now raise the substage condenser (using the
  condenser focusing knob) and focus the image of
  the field diaphragm sharply onto the
  already-focused specimen.
• This image of the field diaphragm should appear
  as a focused polygon.

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Setting Up Köhler illumination

• If the image of the field diaphragm is not
  centered in the field of view, use the condenser
  centering screws (or knobs) to center the image
  of the field diaphragm.
• Then open up the field diaphragm until it just
  disappears from view.

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Setting Up Köhler illumination

• Centering the image of the field diaphragm

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Setting Up Köhler illumination

• As a rule of thumb adjust the iris/apeture
  diaphragm so that it is 2/3 to 3/4 open. This
  setting usually represents the best compromise
  between resolution and contrast.

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Setting Up Köhler illumination

• You have now set up Köhler illumination with the
  10X objective. If you wish to switch to a higher
  power objective, you must again adjust BOTH the
  field and the iris/aperture diaphragms.
• For example, if you switch to the 40X objective,
  you will have to close the field diaphragm
  somewhat and may have to recenter it (looking at
  a smaller area of the specimen).
• You will also have to open up the iris diaphragm
  somewhat (the 40X objective has a higher
  numerical aperture - light-grasping ability -
  than does the 10X objective).
• Thus, every time you change objectives, you must
  adjust both diaphragms in accordance with the
  steps given above.

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Parfocal

• The parfocal distance, that is, the distance from
  the objective shoulder to the specimen, is set at
  45mm for all objectives.
• This means that focusing can be performed quickly
  and easily, with a minimum of fine adjustment,
  even when switching from a low to a high
  magnification objective.

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Resolution

• Related to the wavelength of light used and to
  the numerical aperture of the objective lens.
• R wavelength of light (?)
• 2NA

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Resolution

• Numerical aperture is determined by the
  refractive index of the medium between the
  specimen and the objective and the sine of the
  angle a.
• In the case of dry lenses, n 1 (air), and for
  oil immersion lenses it has a value depending on
  the refractive index of the oil used, usually
  1.515.

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Resolution

• Resolution increases with increasing numerical
  aperture, or decreasing wavelength of light.
• Limit to resolution for an optical microscope is
  0.2µm or 0.25µm.

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Care and Cleaning of your Microscope

• Parts that require cleaning include
• Oculars and condenser
• Objective lenses
• Light source lens and filters located at the base
  of the microscope
• Microscope body including the stage and stage
  clips

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Care and Cleaning of your Microscope

• Cleaning Supplies
• Canned air or duster
• Lint-free tissue
• Lens tissue and/or cotton-tipped sticks stored in
  a dust-free container
• Microscope Cleaning Solution (MCS)

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Suggested cleaning fluids (MCS)

• Distilled water
• Commercial photographic cleaning solutions
• Windex or sparkle
• Ethanol
• Methlylated spirits
• Petroleum ether 85 , Isopropanol 15
• Histoclear/Histolene

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Cleaning the Oculars and Condenser

• Remove slide from the stage blow off dust with
  canned air or air blower.
• Dampen a new cotton-tipped stick or lens tissue
  with MCS.
• Start in the center of the ocular or condenser,
  and spiral to the outside edge while rotating the
  cotton tip.
• Repeat with a new, dry cotton-tipped stick.
• Repeat the above until the view is clear.

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Cleaning the Oculars and Condenser

• Use only new cotton-tipped sticks in a rotating
  fashion when touching the lens surface to avoid
  scratching the lens with any debris that is being
  removed.
• The oculars and the condenser should be cleaned
  by starting at the center and spiraling outward
  while rotating the cotton tip. Be sure to only
  use new clean cotton-tipped sticks to touch the
  lens surface to avoid scratching the lens.

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Objective Care

• Remove/install objectives using both hands.
  Loosely cup with one hand and twist the barrel
  with the other, being very careful not to touch
  the front lens with your fingers. Take extreme
  caution not to drop the objective!
• Never apply strong physical force to an
  objective. To move another objective into
  position, move the rotating turret do not grab
  the objective and pull on it.
• To remove a stuck objective, never use vice-grips
  or a pipe wrench, which can severely damage the
  optics. Attempt to loosen the threads by applying
  a few drops of water to dissolve salts, or an
  oil-dissolving agent if immersion oil is the
  culprit.

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Cleaning the Objectives

• Remove slide from the stage.
• To clean, remove the objective from the turret.
• Dampen a new cotton-tipped stick or lens tissue
  (folded into fourths) with MCS.
• Hold the cotton-tipped stick at a 45 degree angle
  on the objective lens and twirl.
• Repeat with a clean dry cotton-tipped stick.
• Repeat the above until the view is clear.

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Cleaning the Objectives

• The 100x objective may need to be cleaned during
  a microscope session as well as after the session
  if blurry optics are noticed when looking at an
  object on oil.
• Oil objectives should be cleaned after each
  microscope session. Dampen a cotton-tipped stick
  with MCS. Cleaning should be done at a 45 degree
  angle to the objective. Be sure to only use new
  cotton-tipped sticks to touch the objective to
  avoid scratching.

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Cleaning Objectives

• Examine the lens carefully by removing the
  microscopes eyepiece, looking through it
  backwards, and holding it up to the edge of the
  objective, to see a magnified image of the lens.
• Angle the objective so that the room light is
  brightly illuminating the lens surface it should
  appear spotlessly clean. If not, repeat the above
  procedure.
• This is also a good way to examine a lens closely
  for scratches or other imperfections.

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Cleaning Stage, Body, Light Source Lens and
Filters

• Remove slide from the stage.
• Use canned air or blower to remove dust from the
  light source lens, microscope stage and body.
• Use lens paper dampened with MCS to wipe off the
  surface of the light source lens.
• Use lint-free tissue dampened with MCS, to wipe
  off the stage and stage clip remove the stage
  clip if necessary.
• Repeat as needed, making sure to use only new
  lens paper to touch the lens surface to avoid
  scratching.
• Wipe down all surfaces of the microscope body
  with lint-free tissue dampened with MCS.

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Cleaning Stage, Body, Light Source Lens and
Filters

• Use dampened paper towels to clean off the work
  bench area around the microscope after completing
  each day of work. Be sure to use your microscope
  dust cover or clean plastic bag when the
  equipment is not in use.
• The light source lens, microscope stage and body
  should be cleaned every day of use. Use canned
  air to blow dust off the light source lens and
  microscope body.
• Use lens paper dampened with MCS to wipe off the
  surface of the light source lens.
• Use only new lens paper to touch the lens surface
  to avoid scratching.
• Wipe off the stage and stage clip.
• Cover the microscope when the equipment is not in
  use.