🔬 Introduction

• FISH = A technique that uses fluorescently labeled probes (short DNA/RNA sequences, 15–25 nt) to bind ribosomal RNA inside cells.
• Why it’s powerful:
  • Identifies microbes without cultivation.
  • Works directly on complex environmental or medical samples.
  • Visualizes microbes with epifluorescence microscopy or flow cytometry.
• Applications: environmental microbiology, medical microbiology, and microbial ecology (community structure + spatial arrangement).
• 🚨 Limitations:
  • Some cells can’t be permeabilized.
  • Weak signals in cells with low ribosome content.
  • Poor differentiation at strain level.
  • Manual counting is slow and error-prone in biofilms.

🚫 Causes of Missing or Weak Signals & Solutions

1. Accessibility of probe target sites

• rRNA structure blocks probe binding.
• Fix: Helper probes (unlabeled probes that “open up” the RNA for main probe binding).

2. Peptide Nucleic Acids (PNA) 🧬

• Synthetic DNA mimics with neutral backbone → bind more strongly.
• Benefits: Work at higher temps, open rRNA folds, great for Gram-positives & cyanobacteria.
• Downside: Expensive, need custom optimization.

3. Nucleotides as Quenchers

• Nearby nucleotides can reduce fluorescence (especially guanosine).
• Probe design must consider both specificity and fluorescence quenching.

4. Self-ligating probes 🔗

• Two probes bind next to each other and “self-ligate,” removing a quencher.
• Advantage: Only correctly bound probes fluoresce → low background noise.
• Limitation: Requires adjacent binding sites → harder to design.

5. Polyribonucleotide probes

• Multi-labeled probes (via PCR or transcription).
• Much brighter signals, good for slow-growing/oligotrophic microbes.
• ❌ Cons: labor-intensive, sensitive to degradation, less specificity.

6. Enzymatic Signal Amplification (CARD-FISH)

• HRP-labeled probes + tyramide amplification = 🔥 strong signals.
• Especially useful for low-ribosome-content cells.
• Problem: Cell permeabilization steps can kill cells → bias community analysis.
• Fix: embed cells in agarose → less lysis.

📊 Quantification – Moving Towards Automation

• Manual counting = 🐌 slow + inaccurate.
• Alternatives:
  • Flow cytometry (fast, but needs single cells).
  • Digital image analysis (works for aggregates, biofilms).
  • Spike-FISH: add known E. coli as internal standard to calibrate counts.
• Automation with confocal microscopes + motorized stages → high-throughput quantification.

🧬 Clone-FISH & New Targets

• Problem: Probes often fail with uncultured microbes.
• Clone-FISH: Express environmental rRNA genes in E. coli, test probe binding.
  • Helps optimize probes when no pure culture exists.
  • Can be extended to other RNAs (e.g., functional genes like nifH, amoA).
• Other targets:
  • tmRNA (10Sa RNA): involved in protein rescue.
  • Intergenic rRNA spacers: allow species/strain differentiation.

⚡ Inferring Activity & Function

• rRNA content ≠ always activity! Some starved cells still glow strongly.
• Solutions:
  • Target spacer RNAs (short-lived, reflect activity).
  • Combine FISH with BrdU incorporation (DNA synthesis marker).
  • MAR-FISH (Microautoradiography + FISH): track substrate uptake at single-cell level.
    • E.g., who eats acetate, who reduces iron, etc.
• Great for linking identity → function in natural communities.

🏁 Conclusions & Outlook

• FISH is now a robust, rapid, widely used tool for microbial identification and quantification.
• Future needs:
  • Better preservation of 3D structures (biofilms).
  • Improved digital analysis for spatial mapping.
  • Linking microbial neighbors to interactions.

🆕 Update

• Accessibility maps of 16S rRNA for multiple organisms (Bacteria, Archaea, Eukarya) were made.
• Shows probe accessibility depends on phylogenetic relatedness → aids universal probe design.

👉 Quick recap memory hooks:

• Helper probes = sidekicks that open doors.
• PNA = strong synthetic keys.
• Self-ligating probes = 🔒 lock-and-key pairs with built-in check.
• Polyribonucleotide probes = megaphone for quiet microbes.
• CARD-FISH = turbo boost for weak signals.
• MAR-FISH = watch microbes eat in real time 🍽️.