Microscope Notes for Medical Laboratory Technician

Objectives of Using a Brightfield Microscope

• Describe the use of a Brightfield microscope in the clinical lab.
• Identify the parts of the upright Brightfield microscope.
• Explain the theory of Kohler illumination.
• Explain how to focus a slide on the microscope.
• Discuss maintenance, cleaning, and documentation of microscopes.
• Perform Kohler Illumination on a Brightfield microscope.

Description of the Microscope

• A microscope is a magnifying device used primarily in clinical laboratories.
• The compound light microscope, also known as the brightfield microscope, has two main lenses: the objective lens and the ocular (eyepiece).
• Magnification:
  • The total magnification is the product of the objective and ocular lens magnifications:
  • Total Magnification = Magnification of Objective × Magnification of Ocular
  • Example: For a 10× ocular and a 10× objective, Total Magnification = 10 × 10 = 100×.
  • Oculars typically have a fixed magnification of 10x, while objective lenses can vary (4x, 10x, 20x, 50x, 100x).
  • Magnification refers to the increase in size of the image, measured in diameters.

Resolution in Microscopy

• Resolution is the ability to distinguish two closely spaced points as separate entities.
• It represents the limit of usable magnification; exceeding this can lead to "empty magnification".
• The concept of resolving power indicates how much detail can be seen and is affected by lens quality and parasitic factors.

Parts of the Compound/Brightfield Microscope

1. Framework:

   • Base: Firm support structure.
   • Arm: Holds components and serves as a handle.
   • Stage: Platform where specimens are placed; often includes mechanical controls.

2. Oculars (Eyepiece):

   • Usually 10x magnification (with some variations such as 5x or 20x).
   • Binocular microscopes have two ocular lenses, while monocular ones have one.
   • Interpupillary distance and diopter adjustment for focus.

3. Objectives:

   • Primary component for magnification and fine detail observation.
   • Varied magnifications inscribed on lens, often 4x, 10x, 40x, 100x.
   • Parfocal design: minimizes focus adjustment when switching objectives.

4. Focusing System:

   • Coarse Adjustment: Rapid movement for rough focusing.
   • Fine Adjustment: Slow movement for precise focusing.

5. Iris Diaphragm:

   • Controls light intensity passing through the specimen.
   • Position affects resolving power; proper illumination is pivotal for analysis.

Illumination System

• Brightfield Illumination: Most common for laboratory analysis.
• Different illumination methods used for specialized applications:
  • Phase contrast, Darkfield, Fluorescence, Electron microscopy.
• The condenser focuses light onto the specimen and can usually be adjusted for optimal imaging.
  • The position and configuration can also enhance contrast based on specimen type.

Köhler Illumination

• Köhler Illumination: Techniques for even illumination and optimized image quality.
  • Requires adjusting the condenser and diaphragms precisely.
  • Ensures the numerical aperture of the objectives matches that of the condenser.
• Steps for Setting Up:
  1. Place slide on stage and focus with a 10x objective.
  2. Adjust interpupillary distance.
  3. Adjust condenser to achieve proper visibility of diaphragm.
  4. Use field diaphragm to fill the viewing area with light.

Focusing a Slide

• Procedure:
  1. Ensure the microscope is on and a light source is set up.
  2. Load slide onto the stage.
  3. Use coarse adjustment to raise the stage until close to the slide and then focus with fine adjustments.
  4. To switch objectives, use side viewing to adjust the nosepiece without losing focus.

Maintenance and Cleaning

• Follow SOP or manufacturer's instructions.
• Daily checks should include:
  • Clean objectives, proper storage when not in use, and ensure the microscope is turned off.
• Document all maintenance activities for lab accreditation and safety compliance.
• Use lint-free tissue for cleaning lenses, especially oil-immersion objectives after use.

Documentation Practices

• Maintain a record of all cleaning and maintenance actions related to each microscope.
• Create logs to include:
  • Date, activities undertaken, and who performed the task.