IVF Laboratory Technology - Week 1 Microscope Components

Microscope Components

Illumination

  • Modern microscopes typically have an integral light source that is highly controllable.
  • The most common light source is an incandescent tungsten-halogen bulb within a reflective housing.
  • This housing projects light through a collector lens and into the sub-stage condenser.

Tungsten Lamps

  • Tungsten lamps vary in design, with diverse models featuring different envelope shapes, mounting fixtures, and filament arrangements.
  • Figure 3 illustrates a typical selection of tungsten lamps used in optical microscopy.

External Illumination

  • Microscopes lacking an internal light source use external light sources for illumination.
  • Figure 6 illustrates typical external illumination sources, which may contain various incandescent tungsten bulbs.

Sub-stage Condenser and Diaphragm

  • The sub-stage condenser gathers light from the microscope's light source.
  • It concentrates the light into a cone, illuminating the specimen uniformly across the viewfield.
  • Proper adjustment of the condenser light cone is critical to optimize the intensity and angle of light entering the objective front lens.
  • Each time an objective is changed, a corresponding adjustment must be performed on the sub-stage condenser to provide the proper light cone for the numerical aperture of the new objective.

Abbe Condenser

  • A simple two-lens Abbe condenser is illustrated in Figure 1.
  • Light from the illumination source passes through the condenser aperture diaphragm, located at the base of the condenser.
  • Internal lens elements concentrate the light, projecting it through the specimen.

Aperture Diaphragm

  • The size and numerical aperture of the light cone are determined by adjusting the aperture diaphragm.
  • After passing through the specimen, the light diverges into an inverted cone with the correct angle to fill the front lens of the objective.

Specimen Stages

  • All microscopes include a stage where the specimen (usually mounted on a glass slide) is placed for observation.
  • For living specimens, the stage can be fitted with a heating element to maintain a specific temperature.
  • Stages are often equipped with a mechanical device to hold the specimen slide in place and smoothly translate it back and forth and side to side (Figure 1).

Objectives

  • Microscope objectives are the most important components of an optical microscope, determining the quality of images produced.
  • A wide range of objective designs are available.
  • The objective is the most difficult component to design and assemble and is the first component that light encounters as it proceeds from the specimen to the image plane.
  • Objectives are named for their proximity to the object (specimen) being imaged.

Modern Objectives

  • Modern objectives, composed of numerous internal glass lens elements, have reached a high level of quality and performance.
  • The extent of correction for aberrations and flatness of field determines the usefulness and cost of an objective.
  • Figure 1 illustrates a typical 60x plan apochromat objective, including common engravings that contain all specifications necessary to determine its designed use and conditions for proper use.

Eyepieces - Oculars

  • Eyepieces work in combination with microscope objectives to further magnify the intermediate image so that specimen details can be observed.
  • Best results in microscopy require that objectives be used in combination with eyepieces that are appropriate to the correction and type of objective.
  • Eyepieces may have a focus adjustment and a thumbscrew to fix their position.
  • Manufacturers often produce eyepieces with rubber eye-cups that correctly position the eyes from the front lens, and block room light from reflecting off the lens surface and interfering with the view.

Inverted Microscope

  • The optical train is in a "U" shaped configuration.
  • The position of the illumination source, condenser, and objectives are upside down compared to the traditional compound microscope.
  • The illumination source and condenser are above the stage with the optical train projected downward.
  • The objectives and nosepiece are below the specimen stage pointing upward.
  • The binocular head or ocular portion remains in the standard orientation.

Primary Purpose

  • The primary purpose is to view the specimen from below.
  • This design grew from the needs of microscopists to view living specimens.
  • Traditional methods of sandwiching thin sections of tissue between a glass slide and coverslip work well for non-living (fixed) tissues, but do not generally permit observation of living cells.
  • To view living cells or tissues, some type of support system is used to maintain their viability during observation.
  • Typically culture dishes, flasks, or tubes containing a supportive culture medium have been used as in vitro culture vessels.
  • These vessels create a problem as they are too thick for the typical microscope objective to focus through.
  • The inverted microscope was created to allow the cells to be imaged through the relatively thin bottom of these culture vessels.

Disadvantages - Inverted Microscope

  • Cost is a major disadvantage as fewer inverted microscopes are sold leading to less economy of production.

  • More complex and expensive to build.

  • Unlike standard microscopes designed for standardized cover glass thickness, the objectives on the inverted microscope must be able to be used with different containers of various thickness and optical characteristics, requiring correction collars.

  • Special higher power objectives are used with the inverted microscope, corrected for a much longer working distance.

  • These special objectives are designated by some manufactures as "LWD" (long working distance) and "ULWD" (ultra-long working distance) objectives.

  • Limitation on image magnification and quality.

  • The maximum magnification is restricted.

  • $40x$ objectives are usually the highest power objective available.

  • Oil immersion $100x$ objectives are not commonly available.

  • There is a relative reduction of optical clarity and uniformity of the walls of the culture vessels (petri dishes etc) compared to a good quality cover slip.

  • The quality of the image therefore, may not be as good as looking through a conventional microscope with comparable objectives.

Specimen Stages (Inverted Microscope)
  • The main difference is the large stage opening that accommodates an insert on the inverted microscope stage.
  • The inserts (Figure 4) are usually made of stainless steel & have openings of various sizes to allow for large differences in sample size.
  • For instance, with tissue culture research large culture flasks often must scan to observe the entire population of cells and this is much easier to accomplish when the stage insert has a large opening.
Micromanipulators
  • It is often necessary to manipulate the specimen while it is being observed under the microscope.
  • This is the case in many tissue culture and in vitro fertilization experiments, as well as genetic implantation procedures that require close observation of the sample during the experiment.
  • The micromanipulator stage illustrated in Figure 6 is an example of this type of equipment.

Stereomicroscope

Principle

  • Human eyes and brain function together to produce stereoscopic vision, which provides spatial, 3D images.
  • This is due to the brain's interpretation of two slightly different images received from each retina.
  • The stereomicroscope takes advantage of the brain’s ability to perceive depth by transmitting twin images that are inclined by a small angle (usually between 10 and 12 degrees) to yield a true stereoscopic effect.

History

  • The first modern stereomicroscope was introduced in the United States by the American Optical Company in 1957.
  • Named the Cycloptic®, this breakthrough design featured
    • A die-cast aluminium housing
    • A constant working distance (four inches, was one of the longest produced)
    • An internal magnification changer, which allowed the observer to increase the objective magnification from $0.7x$ to $2.5x$ in five steps
  • The Cycloptic had a threaded mount in the lower microscope body to secure the objective into position just beneath a rotatable drum containing two pairs of afocal Galilean-style telescopes.

Types

  • Stereomicroscopes can be roughly divided into two basic families, each of which has both positive and negative characteristics.
    • 1) The oldest stereo-microscope system, named after the inventor Greenough, utilizes twin body tubes that are inclined to produce the stereo effect.
    • 2) A newer system, termed the common main objective, utilizes a single large objective that is shared between a pair of eyepiece tubes and lens systems.

Greenough v CMO

  • Common main objective stereomicroscopes can cost several times more than a Greenough microscope, which is a chief consideration for manufacturers who may require tens to hundreds of microscopes.
  • However, there are exceptions. If a common main objective microscope is the better tool for a job, the true cost of ownership may be lower in the end.Greenough v CMO