Microscopy: Key Concepts and Lab Procedures

Magnification

• Key idea: Magnification makes an image appear larger than its actual size. It is achieved using a series of lenses in the microscope.
• Ocular (eyepiece) lens magnification: 10x.
• Objective lens magnifications:
  • Scanning objective: 4x
  • Low power objective: 10x
  • High-dry objective: 40x
  • Oil immersion lens: 100x
• Total magnification formula:
  • $\text{Total magnification} = \text{eyepiece} \times \text{objective}$
  • Example: using the scanning objective lens
  • $10 \times 4 = 40\times$
• Practical mapping of magnifications:
  • Scanning: $10 \times 4 = 40\times$
  • Low power: $10 \times 10 = 100\times$
  • High-dry: $10 \times 40 = 400\times$
  • Oil immersion: $10 \times 100 = 1000\times$
• Note: Total magnification depends on both the ocular and objective lenses.

Resolution (Resolving Power)

• Definition: The ability to distinguish between two closely adjacent points.
• Affects image clarity; better resolution = crisper image.
• Primary factors:
  • Wavelength of light used: light travels as waves; shorter wavelengths yield better resolution.
  • Numerical aperture of the lens: higher NA gathers more light and improves resolution.
• Visible-light range: $350\ \text{nm} \le \lambda \le 700\ \text{nm}$
• Common practice for good resolution: blue light around $\lambda \approx 480\text{--}500\ \text{nm}$
• Note: For this class, you do not need to calculate numerical aperture; no formulas are required to compute NA.

Working Distance

• Definition: The space between the stage and the objective lens when the specimen is in focus.
• Working distance decreases as magnification increases (roughly).

Parfocal

• Definition: Once the specimen is in focus with one objective, it should remain in approximate focus when switching to another objective.
• Facilitates quicker refocusing during objective changes.

Microscope Parts

• Arm
• Base
• Light source
• Stage
• Stage clips and knobs
• Condenser
• Iris diaphragm
• Nosepiece
• Ocular lens (eyepiece)
• Objective lenses: scanning, low power, high-dry, oil immersion
• Course Adjustment Knob
• Fine Adjustment Knob

Ocular/Eyepiece and Stage Details (as labeled on a typical microscope)

• Portions often labeled: Ocular lens, Stage, Stage clips, Nosepiece, Base, Arm, Light source, Condenser, Iris diaphragm, Course and Fine Adjustment Knobs, Stage adjustment knobs
• Typical magnifications on the objective lenses correspond to the lens you rotate into place (4x, 10x, 40x, 100x with oil immersion)

How to properly focus (step-by-step)

• Always start with the scanning objective lens (4x).
• Bring the stage up as high as it will go.
• Looking through the ocular, slowly move the stage down using the coarse adjustment knob.
• Stop when you see color (blue, green, pink, or purple). If you see gray, you are likely looking at trash.
• Use the fine adjustment knob to bring the colored object into focus.
• Change to the low-power objective lens using the nosepiece.
• Use the coarse and fine adjustment knobs to refocus the colored object.
• Change to the high-dry objective lens with the nosepiece.
• Use the coarse and fine adjustment knobs to refocus again.

Oil immersion procedure (for viewing bacteria)

• Prerequisite: The object must be in focus on the high-dry lens before using oil immersion.
• Move the nosepiece halfway between the high-dry and immersion oil.
• Add a drop of immersion oil to the slide.
• Move the nosepiece to click the oil immersion lens into place.
• Important: The oil immersion lens will touch the oil; it must touch the oil for proper imaging.
• Do not move the stage downward during this process.
• Use only the fine adjustment knob to focus the object when using oil immersion.
• After viewing, clean the oil off the slide and off the lens with lens paper (not brown paper towels).

Video resource

• There is a short video (about 5 ½ minutes) showing proper focusing and oil immersion use: https://www.youtube.com/watch?v=_DZ XqPZ7aTc
• Note: The link contains a space due to transcription; if copying, use the full correct URL without spaces.

Lab 2 Practice: What you will view

• Slides to view: several bacterial slides, some cyanobacteria, and one yeast slide (as described in the Materials heading of the lab book, page 12).
• Bacteria viewing requirements:
  • Must use the oil immersion lens with total magnification of 1000x to see bacteria.
  • Common bacterial shapes:
  • Coccus/Cocci (spherical)
  • Bacillus/Bacilli (rod-shaped)
  • Spirillum/Spirilli (spiral-shaped)
• Examples seen on slides:
  • Streptococcus (chains of cocci)
  • Staphylococcus (grape-like clusters of cocci)

Bacterial Shapes (quick reference)

• Bacili (Bacillus/Bacilli): rod-shaped
• Cocci: spherical
• Spirilli (Spirilli): spiral-shaped

Cyanobacteria and Yeast

• Cyanobacteria:
  • Larger than some bacteria and do not require oil immersion; high-dry objective is sufficient.
  • They are photosynthetic; make their own food like plants.
  • They are bacteria in a broad sense but do not conform to the three classic shapes (cocci, bacilli, spirilli).
• Yeast:
  • Single-celled fungus
  • Commonly used to bake bread and brew beer/wine

Additional notes and practical tips

• The imaging color cues help verify focusing: colored objects indicate you are near focus; gray indicates you may be viewing debris.
• The surface quality of lenses affects light gathering; scratches, bumps, or bubbles reduce resolution.
• Do not worry about calculating numerical aperture in this course; focus on understanding the concept and how resolution relates to wavelength and NA conceptually.
• Always handle slides and lenses with care; use lens paper for cleaning, never rough towels.

Key numerical references

• Visible light wavelength range: $350\ \text{nm} \le \lambda \le 700\ \text{nm}$
• Blue light commonly used for better resolution: $\lambda \approx 480\text{--}500\ \text{nm}$
• Magnification cheatsheet:
  • Scanning objective (4x) with ocular (10x): $10 \times 4 = 40\times$
  • Low power objective (10x) with ocular (10x): $10 \times 10 = 100\times$
  • High-dry objective (40x) with ocular (10x): $10 \times 40 = 400\times$
  • Oil immersion (100x) with ocular (10x): $10 \times 100 = 1000\times$
• Total magnification is the product of the ocular and objective magnifications.