The Microscope

The microscope is an essential tool to the hematology laboratory professional. It is a piece of equipment that is stylistically simple in design, yet extraordinarily complex in its ability to magnify an image, provide visual details of that image, and make the image visible to the human eye.2 Most commonly used today are compound microscopes, which use two lens systems to magnify the image. The ocular devices on the microscope provide an initial X10 magnification, and then additional magnification is obtained through the use of three or four different powered objectives.3 A light source is located within the microscope base. Light is beamed to the image directly, or filters are used that vary the wavelength. In addition, a diaphragm apparatus is usually located in the base of the microscope. Opening or closing the diaphragm can optimize or reduce the volume of light directed toward the image.4 This is most useful when examining cellular structures in the nucleus that need more light to be properly visualized. Below is a brief description of the most significant parts of the microscope

Significant Parts of the Microscope

The eyepieces, or oculars, are located laterally to the microscope base (Fig. 1.1) and function as an additional magnification component to the objective magnification. Most microscopes are binocular and contain two eyepieces, each of which will magnify the diameter of an object placed on the stage to the power of the eyepiece, usually X 10.

The objectives of the compound microscope are X10, X40, or X100. Often, a X50 (oil) magnification will be incorporated. Each objective has three numbers inscribed on the objective: a magnification number, an aperture number (NA), and a tube length number. The NA refers to the resolution power of the objective, the ability of the objective to gather light. The higher the NA number, the higher is the resolution. Tube length refers to the distance from the eyepiece to the objective. Magnification refers to how large the image will appear, as well as how much of the viewing field will be observed. Objectives on modern microscopes are com

- Eye piece

Adjust to eye width Move adjustable eye piece for optimal focus

Figure 1.1 Compound microscope.

- Eye piece

Adjust to eye width Move adjustable eye piece for optimal focus

Figure 1.1 Compound microscope.

Stage Secures glass slides

Stage adjustment knobs Move stage left to right or front to back

Base

Iris diaphragm Left to right to maximize or minimize light

Light source

Objectives 10x = Low power 40x = high dry 100x = Oil immersion

Stage Secures glass slides

Iris diaphragm Left to right to maximize or minimize light

Course adjustment Use for preliminary focus

Fine adjustment Use for fine tuning

Stage adjustment knobs Move stage left to right or front to back

Base

Light source posed of many lenses and prisms that produce an extremely high quality of optical performance.

The iris diaphragm, located below the microscope stage, increases or decreases light from the microscope light source. If the diaphragm is opened to its full capacity, the cell or structure is viewed with maximum light. If the diaphragm is minimally opened, the cell or structure is much less illuminated, which may be desirable depending on the source of the sample (i.e., hematological cells versus urine casts).

The stage is a flat surface with an opening created for light to pass through. Two flat metal clips have been mounted in which to secure the glass slide. Below the stage surface are two control knobs that move the slide in a horizontal or vertical fashion.

Coarse and fine adjustment knobs are located on either side of the microscope base. These adjustments bring the image into focus through movement of the stage, which is either raised or lowered according to the level of focus needed.

Care of the Microscope

The microscope is an essential piece of equipment to the practice of hematology and must be handled with care and respect. Hematology instructors owe it to themselves to teach the care and maintenance of the microscope in hopes that these "best practices" can be adopted and practiced in the workplace. The microscope should be on a level, vibration-free surface. If it needs to be lifted from a storage cabinet to another location, the microscope must be secured on the bottom by one hand and held by the neck with the other hand. Additionally, users must be instructed on how to move

6 Part I • Basic Hematology Principles objectives from one position to another without dragging non-oil objectives into oil from a slide left on the stage. The high-dry objectives must never be used with oil, only with coverslipped slides. Objectives are easily scratched or damaged by careless handlers; consequently, they must be cleaned with lens paper after each use. Oil objectives should be wiped free of oil when not in use, and eyepieces must be cleaned with lens paper from dust, dirt, or cosmetic debris with each viewing. Good microscopy habits should always be cultivated, practiced, and communicated. Microscopy guidelines should be posted in each area where microscopes are used. The guidelines should include

• General use of the microscope

• Instructions for transporting the microscope

• Instructions for proper cleaning of the microscope

• Storage guidelines that include proper position of microscope cording

Corrective Actions in Light Microscopy

Many of the problems that are encountered when using a microscope can be easily corrected by using common sense. Some of the most common "problems" in light microscopy are as follows:

• Image cannot be seen at any power—Try turning the slide over; perhaps the wrong side of the slide has been placed on the microscope stage.

• Fine details cannot be detected in immature cells—For immature cells, use the X100 lens and open up both diaphragms to the maximum width, for maximum light.

• The X40 objective is blurred—Try wiping off the X100 lens; perhaps the X100 lens was oil filled and was dragged across the slide.

• Dustlike particles appear on the slide but they are not large enough to be platelets— Perhaps mascara has been left on the eyepiece; use lens cleaner to clean the eyepieces.

Innovations in Microscopy

Digital microscopy is gaining in popularity as a routine piece of equipment in hematology laboratories. Simply stated, these microscopes scan blood smears for cells, identify them, calculate a white blood cell differential count, and then store the cellular images of the cells for future review. Slides are then reviewed for red cell morphology and abnormalities by a trained operator.5 The initial purchase cost is expensive, yet the speed, sensi tivity, and reduced technologist time are making this an attractive option for larger laboratories.

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