🔬 Introduction to Microscopy

Microscopy allows us to visualize structures too small for the naked eye. Different microscopes are designed for different purposes — some excel at speed, others at depth, resolution, or contrast.

🧪 Microscopy Session Setup

• Students are introduced to two microscope types available:
  • Scanning Laser Confocal Microscope
  • Spinning Disk Confocal Microscope
• The purpose: to understand how each microscope works, how images are captured, and how signals (numbers in a digital picture) are converted into meaningful visual data for analysis.

⚙️ Basic Principle of Microscopy

🕳️ What happens when you magnify something?

• You start with a sample (specimen).
• Light is shone through or onto it.
• The objective lens bends (refracts) the light, focusing it to form an image.
• The eyepiece further enlarges this image.
• You’re not actually seeing the specimen itself — you’re seeing an optical projection (mirror image) created by refocused light.

💡 Magnification ≠ amplification of signal — it’s simply making the same optical information appear larger.

🌈 Types of Microscopy

1. Light Microscopy

• Uses visible light to illuminate the sample.
• Works best for samples with natural contrast (e.g., stained cells, thick tissues).
• Limited by the wavelength of light — typically allows resolution down to around 200 nm.

2. Fluorescence Microscopy ✨

• Instead of using white light, UV or specific wavelengths excite fluorescent molecules.
• These molecules emit light at longer wavelengths, producing bright images against dark backgrounds.
• Allows for specific labeling of cellular components (e.g., GFP-tagged proteins).

3. Confocal Microscopy

There are two main types discussed:

🔁 Scanning Laser Confocal

• Uses a laser beam that scans the sample point by point.
• Provides high-resolution and optical sectioning (3D reconstruction possible).
• Slower because it scans sequentially.

💿 Spinning Disk Confocal

• Uses many tiny pinholes on a rotating disk to scan multiple points at once.
• Much faster, ideal for live-cell imaging.
• Slightly lower contrast than scanning laser systems.

4. Expansion Microscopy 🧼

• A technique where the sample is physically expanded (embedded in a swellable gel).
• Enables high-resolution imaging with standard microscopes.
• Applied in research to visualize subcellular structures that would otherwise require super-resolution systems.

5. Advanced and Specialized Microscopy Types

These push beyond standard optical limits:

Resolution improves as wavelength decreases — electrons have much shorter wavelengths than light.

📏 Resolution and Scale

Microscope resolution depends on the wavelength of the light source:

• 👁️ Naked eye: ~0.1 mm (e.g., a child’s height, hands, hair).
• 🔬 Light microscope: ~200 nm (e.g., cells, bacteria).
• ⚡ Electron microscope: ~0.1 nm (e.g., viruses, proteins, atoms).

The shorter the wavelength, the higher the resolving power.

🧠 How Contrast Works

Contrast makes structures visible. Without it, transparent cells appear flat and featureless. Different techniques enhance contrast in unique ways.

1. DIC (Differential Interference Contrast) ⚔️

• Uses polarized light.
• A prism splits the light beam into two slightly offset rays.
• When recombined, interference patterns create areas of light and dark.
• Produces a 3D-like shadowed image that reveals fine structures.

💡 Fun fact: In DIC, your perception can flip — what looks like a bump can appear as a hole if your brain reverses the contrast. You can “blink” to see this optical illusion.

2. Phase Contrast Microscopy 🌗

• Converts differences in refractive index into brightness variations.
• Great for live, unstained cells — shows internal structures clearly.

3. Fluorescence Staining 🎨

• Adds fluorescent dyes or genetically encoded markers to highlight specific structures.
• Each fluorophore emits a unique color, allowing multi-label imaging (e.g., nucleus blue, actin green).

🖼️ From Image to Data

When capturing an image:

• The microscope collects light intensity values (numerical data).
• These are converted into digital pixels.
• Image analysis tools can then quantify:
  • Signal intensity
  • Shape, size, and localization of structures
  • Co-localization between channels

This transforms microscopy from qualitative (pretty pictures) to quantitative (scientific measurements).

🧩 Summary Table

🧬 Bonus: Upcoming Guest Lecture Mention

At the end, the file mentions a guest lecturer from Aarhus University, who studies evolution and the origin of life and has written a Danish book about it (“Da livet opstod” — When Life Arose).