🧠 Overall theme

Goal: Learn how to visualize and identify microbes and study their function and activity using molecular imaging.

🔬 1. Fluorescence In Situ Hybridization (FISH)

• Concept: Uses short fluorescent DNA probes that bind to complementary rRNA sequences inside fixed cells.
• Purpose: Identify and visualize microorganisms directly in environmental samples — no culturing needed.
• Probes: Usually 15–20 bases long, designed to target conserved rRNA regions.
• Fluorescence: Each probe carries a fluorescent dye (e.g., green, red, blue) 💚❤️💙 so multiple taxa can be detected simultaneously.

🧩 2. Probe specificity and hybridization

• DNA probe binds only if base pairing matches the rRNA sequence.
• Binding strength depends on:
  • Temperature 🌡️
  • Ion strength (e.g., NaCl concentration)
  • Formamide (used to fine-tune stringency)
• Mechanism: Formamide competes for H-bonds between nucleotides, controlling how tightly the probe binds.

💡 3. Probe labeling methods

• Direct labeling: Fluorescent molecule (e.g., Cy3, FITC) attached to probe end (5′ or 3′).
• Indirect labeling: Probe tagged with an enzyme or molecule (e.g., DIG, HRP) → detected via color or signal amplification (TSA = Tyramide Signal Amplification).
• Goal: Get a strong, specific fluorescent signal.

🌈 4. Typical probe examples

Result → Multicolor FISH images where each group glows differently.

🧠 5. Confocal Laser Scanning Microscopy (CLSM)

• Uses laser light focused through a pinhole to capture sharp, 3D optical sections.
• Principle: Laser excites fluorophores → emitted light passes through pinhole → detected by photodetector.
• Advantage: Eliminates out-of-focus blur → produces clear 3D reconstructions 🧱✨.

🧫 6. Comparing imaging methods

• Epifluorescence microscopy: Quick overview, more background noise.
• CLSM: Precise 3D view of spatial structure (e.g., biofilms).
• Used to visualize complex microbial communities — showing where sulfate or iron reducers live in biofilms.

🌍 7. Application: Biofilm communities

• Biofilms contain layers of different microbial types:
  • Heterotrophs on the surface.
  • Autotrophs deeper inside.
• FISH + microscopy reveals spatial organization = who lives where and possibly why.

🧬 8. Probe hierarchy (“top-to-bottom” approach)

Start broad → zoom in:

1. Kingdom-specific probe (e.g., Bacteria, Archaea)
2. Group-specific
3. Order-specific
4. Species-specific Used on fixed samples (PFA for Gram-negative, ethanol for Gram-positive).

🎨 9. Image analysis

Quantitative information from FISH images:

• Cell or colony morphology
• Fluorescence intensity
• Counting organisms (number or biomass)
• Co-localization of taxa
• RGB color fusion to overlay different fluorophores 🎨 → Enables numerical analysis, not just pretty pictures.

☢️ 10. Microautoradiography (MAR)

• Combines radioactive substrate uptake with microscopy:
  1. Cells incubate with a radioactive compound.
  2. Silver grains on film mark metabolically active cells.
• Purpose: Identify which microbes are active or metabolizing a specific substrate.

🔗 11. MAR-FISH combo

• FISH: identifies which species are present.
• MAR: shows which ones are metabolically active.
• Overlay (MAR-FISH) → activity can be linked to identity! 💥

Example: tracking substrate uptake by Meganema in activated sludge → reveals cell-specific activity distributions (Pareto principle: few cells do most of the work).

💎 12. Raman Microscopy / Raman-FISH

• When laser light hits molecules, most light scatters normally, but a small fraction shifts in wavelength (the Raman effect).
• This shift reveals molecular vibrations, creating a unique fingerprint of chemical bonds.
• Advantages:
  • Non-destructive 🌿
  • Can detect isotopic labeling (e.g., H₂O → D₂O)
• Application: Identify and measure metabolic activity of uncultured microbes, like methanogens in biogas sludge, by detecting D₂O incorporation.

🧪 13. Integrating techniques

FISH + MAR + Raman = powerful toolset to:

• Visualize identity 🧫
• Quantify activity ⚡
• Understand metabolism and community function 🌎