🧫 Why Imaging Still Matters in the Genomics Era

Even though DNA sequencing tells us what microbes are present, imaging tells us where and how they live.

• DNA sequencing destroys the sample, so we lose spatial context 🧬❌.
• Imaging keeps 3D structure — we can see how microorganisms interact, form colonies, or organize in biofilms.
• Together, sequencing and imaging are complementary — sequencing gives diversity, imaging gives structure.

🔬 Traditional Microscopy vs Advanced Imaging

Light Microscopy

• Around for 50+ years.
• Resolution: ~1 µm (about the size of a bacterium).
• We can see bacteria but not details inside them.
• High-resolution pictures you see online are computer-generated, not true microscopic images.

Electron Microscopy

• Better resolution, but samples must be fixed and dehydrated.
• This collapses cells — not suitable for living morphology.

So for ecological samples, we need something in between: Fluorescence In Situ Hybridization (FISH).

🐠 FISH = Fluorescence In Situ Hybridization

FISH is not about real fish — it’s a molecular imaging method.

Principle:

We use short DNA probes (15–20 bases) tagged with fluorescent markers (fluorochromes) to bind specific RNA sequences (usually ribosomal RNA) inside cells.

Steps:

1. Embed microbial cells in a matrix.
2. Add fluorescent gene probes.
3. Probes enter cells and bind complementary RNA sequences (usually 16S rRNA).
4. Wash away unbound probes.
5. Observe under a fluorescence microscope — cells that match the probe glow in specific colors ✨.

🧩 Why rRNA Is the Target

• Ribosomes are abundant — up to 75,000 per bacterial cell 😮.
• More ribosomes → brighter fluorescence → easier detection.
• Starved or slow-growing cells have fewer ribosomes → dimmer signal.

⚛️ Hybridization Specificity

Base Pairing:

• G–C: 3 hydrogen bonds
• A–U: 2 hydrogen bonds

Even one mismatch can still bind weakly, causing false positives. To fix this, we tune hybridization conditions so only perfect matches bind tightly.

Instead of temperature (like PCR), FISH uses:

• Formamide concentration to control stringency.
• More formamide → weaker binding → only perfect matches remain paired.

Formamide fits between base pairs like a “molecular wedge,” letting scientists fine-tune probe specificity 🧪.

💡 Direct vs Indirect Labelling

1. Direct FISH

• Fluorescent dye (e.g., FITC, Cy3, Cy5) attached directly to the DNA probe.
• Simple and small — can penetrate cell membranes.
• Limited brightness because only a few fluorophores fit.

2. Indirect FISH

• Probe carries a reporter molecule (like biotin or a small enzyme).
• Later, an antibody or enzyme binds to this reporter, carrying many fluorophores.
• Stronger signal, but bulkier and harder to get inside cells.

Variants:

• CARD-FISH (Catalyzed Reporter Deposition): uses peroxidase enzyme → amplifies fluorescence 💥.
• Allows detection of low-ribosome, slow-growing cells.

🔭 Fluorescence Microscopy Systems

Epifluorescence Microscope

• Light source (often mercury lamp) → beam splitter → sample → detector.
• Detects both in-focus and out-of-focus light → blurry image.
• Detector: CCD camera like in phones 📷.

Confocal Laser Scanning Microscope (CLSM)

• Adds pinhole filters to remove out-of-focus light.
• Uses laser excitation for higher intensity.
• Produces crisp, slice-by-slice optical sections (z-stacks).
• Cost: 💰 5–8 million DKK (vs. ~300–400k for epifluorescence).

Result: clearer, layered 3D reconstructions of microbial structures 🧱.

🌿 Sample Types and Signal Strength

👉 Solution: use signal amplification (indirect FISH) for weak cells.

🧫 Example: Biofilm Structure (Air Filter System)

Biofilm cross-section shows clear microbial zoning:

1. Surface layer — heterotrophic bacteria (eat organic material from air) 🍃
2. Deeper layer — autotrophic bacteria (use CO₂) 🪨
   • Includes ammonia-oxidizing and nitrite-oxidizing bacteria (nitrification).

FISH reveals who lives where — sequencing alone can’t show spatial layering.

🧠 Probe Selection: The Top-to-Bottom Approach

When facing a new sample with hundreds of possible probes:

1. Start broad (e.g., domain or phylum-level probes).
2. Narrow down step by step:
   • Family-level → Genus-level → Species-level.
3. Save time by targeting only relevant groups at each level 🔍.

🎨 Color Coding and Interpretation

Using three probes (Red, Green, Blue):

• Red + Green → Yellow
• Green + Blue → Cyan
• Red + Blue → Magenta
• All three → White → 3 probes = 7 color combinations (2³ − 1 = 7 possible taxa).

🧪 Famous example: Jiri Schneider’s 1999 Munich FISH image — showed all 7 combinations in one field after two weeks of searching!

📈 Quantitative Imaging

We can measure:

• Cell morphology (shape, size)
• Colony structure (clusters, filaments)
• Activity (brighter = more ribosomes = higher metabolic rate)
• Abundance (via image analysis software counting colored pixels → cell counts)

⚠️ But note: large cells = more pixels → apparent bias unless normalized.

💬 Key Takeaways

• FISH bridges the gap between molecular data and spatial organization.
• It identifies and locates specific microorganisms in situ.
• DNA sequencing = who is there 🧬 Imaging = where they are and what they’re doing 🔬
• Combining both gives the most complete ecological picture.