Microscopy Basics for Beginners

Optical Limits • Resolution • Stains • Objectives • Sample Preparation

Light microscopy is one of the simplest and most powerful tools for studying cells.
This guide introduces the essential concepts a beginner needs to understand what can (and cannot) be seen under a standard optical microscope.

This page is designed for beginners working with onion cells, yeast cells, or simple biological samples at home.

1. What a Light Microscope Can and Cannot See

A standard light microscope uses visible light (wavelength ~400–700 nm).
This imposes physical limits on resolution.

1.1. The Resolution Limit

The smallest distance two objects can be distinguished as separate is:

~200 nanometers (0.2 µm)

Anything smaller appears as a blur or becomes invisible.

Visible (usually):

• Entire cells (5–200 µm)
• Nucleus (large plant/animal cells)
• Cell walls (plants, fungi)
• Vacuoles
• Chloroplasts (in green tissues)
• Yeast budding

Not visible (too small):

• DNA (2 nm)
• Genes
• Chromatin structure
• RNA
• Ribosomes (~20–30 nm)
• Mitochondria (barely 0.5–1 µm → too small for details)
• ER, Golgi, vesicles
• Proteins (5–10 nm)

2. Optical Components and Their Purpose

Understanding microscope parts helps you get the best image.

2.1. Objectives

Common magnifications:

• 4× – overview
• 10× – general observation
• 40× – high detail for large cells
• 100× oil – used with immersion oil; high resolution but small field of view

2.2. Numerical Aperture (NA)

NA determines resolving power. Higher NA = more detail.

Typical NA:

• 10× objective: NA 0.25
• 40× objective: NA 0.65
• 100× oil: NA 1.25–1.40

To resolve small structures (yeast, fine cell walls), higher NA is essential.

2.3. Condenser

Focuses light onto your sample.
For best results:

• Use higher condenser position for 40× and 100×
• Adjust iris diaphragm to optimize contrast

2.4. Oil Immersion

Used with 100× objectives only.
The oil reduces light refraction, increasing resolution beyond what air allows.

3. Sample Preparation Basics

Preparing the sample correctly often matters more than the microscope itself.

3.1. Wet Mount (most common)

1. Place a drop of water on the slide
2. Add your sample
3. Place a coverslip at 45° to avoid bubbles
4. Remove excess water with paper

Useful for:

• Onion epidermis
• Yeast cultures
• Pond water
• Plant leaves

3.2. Staining

Stains increase contrast and highlight structures.

Common stains for beginners:

• Iodine → enhances nuclei and starch granules
• Methylene blue → stains nuclei (animal/yeast)
• Safranin → stains plant cell walls
• Crystal violet → stains bacteria and yeast

Onion example:

• Stained nucleus becomes dark purple or brown
• Vacuole remains clear
• Cell wall becomes sharp and visible

Yeast example:

• Staining highlights budding cells
• Nucleus not visible (too small)

3.3. Sectioning (for thicker specimens)

Sharp razor or microtome slice → thin enough for light to pass.

4. Practical Tips for Better Imaging

4.1. Start low → go high

• Begin at 4× or 10× to find your sample
• Switch to 40× for detail
• Only use 100× oil when necessary

4.2. Adjust lighting

• Lower light → increases contrast
• Higher light → increases brightness but reduces contrast

4.3. Clean the optics

Use lens paper ONLY.

4.4. Use proper focus technique

• Coarse focus → low magnification
• Fine focus → medium & high magnification

4.5. Let your sample rest

Air bubbles dissipate after 1–2 minutes.

5. What You Can See in Onion Cells vs. Yeast Cells

5.1. Onion Epidermal Cells (large plant cells)

Very visible:

• Cell walls
• Cytoplasm
• Vacuole
• Nucleus (especially stained)

Not visible:

• Organelles <200 nm
• Mitochondria
• ER/Golgi
• Ribosomes
• DNA

Great sample for beginners.

5.2. Yeast Cells (Saccharomyces cerevisiae)

Visible:

• Round or oval cell shape
• Cell wall
• Budding daughter cells
• Bud scars (with strong light or staining)

Not visible:

• Nucleus
• Chromosomes
• Organelles
• Mitochondria
• Proteins

Yeast are only ~5–8 µm → near the resolution limit.

6. Optical Microscope vs. Fluorescence vs. Electron Microscopy

6.1. Light Microscope

• Resolution: ~200 nm
• Ideal for large cells
• Cannot see molecules

6.2. Fluorescence Microscopy

Uses glowing dyes.
Can see:

• Nuclei in yeast
• Mitochondria
• Actin fibers
• Specific proteins (tagged with GFP)

Requires expensive equipment.

6.3. Electron Microscopy (TEM/SEM)

Resolution: ~0.1–1 nm
Can see:

• Ribosomes
• Mitochondria structure
• Membrane proteins
• Viruses
• DNA fibers

Not available in a home lab.

7. Summary Table: Visibility of Common Structures

8. Quick Beginner Checklist

• Always start at low magnification
• Adjust condenser and diaphragm for contrast
• Use stains to reveal structures
• Work with thin samples
• Know the resolution limit: nothing below ~200 nm is visible