Microscopy Lecture Notes

Microscopy Notes

1st Microscope: The earliest known microscope was developed to observe small objects and living cells.
Anton von Leeuwenhoek:
- Often referred to as the "father of microscopy."
- Notable contributions include documenting the microscopic appearance of:
- Muscle Fibers
- Spermatozoa
- Red Blood Cells
- Blood Flow in Capillaries
- Coined the term "Animalcules" for the cells he observed at that time.

Ocular Lens:
- Magnifies the specimen by an additional factor of 10x.
Arm:
- Supports the entire microscope setup.
Rotating Nosepiece:
- Allows for the changing of the objective lens positioned over the sample.
Stage and Stage Clips:
- Hold the microscope slide in place during focusing and viewing of the specimen.
Condenser:
- Focuses light from the light source through the stage to illuminate the specimen.
Objective Lens:
- Provides magnification to the image by a specific amount.
Rheostat:
- Adjusts the brightness of the light source for better visibility.
Fine and Coarse Focus Adjustment:
- Coarse Focus: Moves the stage up and down in large increments to help initially bring the specimen into focus.
- Fine Focus: Allows for small adjustments to clarify the image focus.
Diaphragm:
- Controls the amount of light that goes through the condenser.
Light Source:
- Provides the necessary illumination for observing the specimen.

Definition: Light microscopy uses visible light to make specimens observable.
Simple Compound Microscopes:
- Composed of multiple lenses.
- Characteristic of being parfocal, meaning switching objective lenses does not result in a loss of focus.
Light Path:
- Light from the source is focused through the condenser lens onto the specimen on the stage.
- The image is subsequently magnified by both the objective lens and the ocular lens.

Total Magnification: Computed by multiplying the magnifying powers of the two lenses used:
- Eyepiece (ocular): Usually 10x or 15x.
- Objectives: Typically include magnifications of 4x, 10x, 40x, and 100x.
- Example:
- A 10x ocular and a 100x objective results in a total magnification of:
  - 10X imes 100X = 1,000X

Definition: Resolution is the ability to distinguish two items as separate units, especially when they are close together.
Challenges in Resolution:
- From a distance, small objects may appear as a single object if resolution is insufficient.
Enhancements to Resolution:
- Abbe Condenser: Harnesses and increases the light beams from the source to allowed more light throughput.
- Iris/Diaphragm: Adjusts the amount of light entering the condenser light source.
- Oil Immersion Technique: Use of immersion oil prevents diffraction or scattering of light, enhancing resolution.
Comparison:
- With Immersion Oil: Retains light rays effectively.
- Without Immersion Oil: Light rays can be lost due to diffraction.

As magnification increases, the area visible through the microscope decreases.
Less light is available in smaller areas, necessitating additional light to maintain resolution.
Adjustments Needed:
- Use immersion oil, condenser, and adjust the iris/diaphragm, which must be opened more widely with increased magnification.

Ensuring the microscope is clean is crucial; each student holds the responsibility for proper maintenance.
Lenses: Check for oil or dirt; clean with lens paper (not bibulous paper or kimwipes).
Immersion Oil: Only oil immersion lens should come into contact with oil; clean other surfaces with alcohol and a kimwipe.

Turn on the light; set to the brightest setting, adjust the iris diaphragm to the 10x setting.
Position the 10x objective lens.
Place the slide on the stage, securing it with stage clips.
Use the coarse adjustment knob to raise the stage to its highest position.
Look through the ocular lenses, slowly lower the stage with the coarse adjustment until you see the specimen.
Adjust image clarity with the coarse adjustment knob.
Switch to the 40x objective lens, then adjust the iris/diaphragm for more light.
Use the fine adjustment knob for the clearest image.
Change to the 100x objective, adjusting it while applying a drop of immersion oil to the slide.
Finalize focus with the fine adjustment knob and iris/diaphragm settings.

Fuzzy Image:
- Possible causes:
- Dirty lenses; clean them with alcohol and lens paper.
- Incorrect use of oil for the 100x lens; ensure oil is utilized properly.
- Light settings: Higher magnifications require more light, adjust using a rheostat or the iris/diaphragm.
Specimen Disappeared:
- Ensure the initial focusing was on the specimen.
- If you lost focus, revert to the 10x lens to relocate the specimen.
- Check that the specimen is centered when increasing magnification; use slide adjustment knobs if needed.
- Confirm that the slide is not upside down, which could lead to disappearance at 100x.
- If you see black or half-black images, ensure the objective lens is completely engaged in position.

Bacterial Morphology:
- Bacteria demonstrate various morphologies, emphasizing the following shapes:
- Cocci: Spherical shape.
- Bacilli: Rod-shaped.
- Spirochete or Spirilli: Spiral shape.
- Bacteria can also form arrangements, large groups of cells interacting together.

Familiarize yourself with the microscope's components and usage steps before staining in subsequent sessions.
Aim to observe and analyze as many prepared slides as possible during your practice.
Document findings by drawing or utilizing photography to capture specimens.
Practice identifying bacterial morphologies and cell arrangements prior to departure.
Ensure to thoroughly clean the microscope and surrounding area after use.