21. Co-localization of two fluorescently labelled proteins in an organelle in cells is usually visualized by:
A. Interference-contrast microscopy
B. Scanning electron microscopy
C. Confocal microscopy
D. Atomic force microscopy
Confocal microscopy eliminates out-of-focus light via pinhole optics, enabling precise 3D co-localization of two fluorescently labeled proteins within organelles.
Correct Answer
C. Confocal microscopy. Provides optical sectioning essential for accurate yellow (co-localized) pixel detection in dual-color imaging.
Option Breakdown
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A. Interference-contrast microscopy: Incorrect. DIC enhances contrast via phase gradients; cannot separate multiple fluorophores or provide z-resolution.
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B. Scanning electron microscopy: Incorrect. Surface topography imaging; no fluorescence detection capability for protein labeling.
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C. Confocal microscopy: Correct. Pinhole rejects out-of-focus fluorescence, enabling true co-localization (yellow pixels) vs bleed-through artifacts.
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D. Atomic force microscopy: Incorrect. Nanoscale surface topography; no optical/fluorescence capability for intracellular protein detection.
Confocal microscopy protein co-localization reveals if GFP-mitochondria and RFP-TOM20 occupy identical 3D space within cells.
Pinhole principle: ~0.75 μm z-resolution eliminates bleed-through; true yellow pixels indicate molecular proximity (<200 nm diffraction limit).
Technique Comparison
| Technique | Fluorescence | Z-Resolution | Co-Localization | Organelle Imaging |
|---|---|---|---|---|
| Confocal | Yes | 0.75 μm | True (pinhole) | Perfect |
| DIC | No | Poor | No multi-color | Contrast only |
| SEM | No | Surface only | No fluorescence | Topography |
| AFM | No | Surface scan | No proteins | Nanoscale |
Co-localization metrics: Pearson’s coefficient >0.8, Mander’s overlap >0.9 indicate true organelle residency.
Applications
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ER-mitochondria contact sites: GFP-IP3R + mito-DsRed
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Golgi recycling: Rab6-GFP + TGN38-RFP
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Lysosomal fusion: LAMP1-GFP + Cathepsin-RFP
Exam Key: Confocal = optical sectioning + multi-color. Others lack fluorescence OR z-resolution.
