22. In resting cells, proteins X and Y are localized in the cytosol. Upon stimulation with lipopolysaccharide (LPS), both of them are phosphorylated and translocate to the nucleus.
You have used antibodies against phosphorylated form of proteins X and Y which are conjugated to either red or green fluorophore.
Keeping optical aberration of lens in mind, which one of the following will be the best for visualizing X and Y in the nucleus by fluorescence microscopy?
A. Anti-green X and anti-red Y
B. Anti-red X and anti-green Y
C. A and B both
D. Anti-blue X and anti-green Y
For nuclear imaging of two phosphorylated proteins X and Y, red and green fluorophores are optimal. Red fluorophores (e.g., Cy5, 650-670 nm) suffer less chromatic aberration than blue (405-450 nm) in standard objectives.
Correct Answer
C. A and B both. Both red-green combinations minimize optical aberration compared to blue-green pairing.
Option Breakdown
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A. Anti-green X and anti-red Y: Correct. Green (FITC, 488-525 nm) + Red (TRITC/Cy3, 550-600 nm) = standard DAPI-free nuclear imaging pair.
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B. Anti-red X and anti-green Y: Correct. Same fluorophores, swapped channels – equivalent performance.
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C. A and B both: Correct answer. Red-green avoids blue channel’s severe chromatic aberration in nucleus visualization.
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D. Anti-blue X and anti-green Y: Incorrect. Blue (DAPI/CFP, 405-460 nm) has highest spherical/chromatic aberration through glass coverslips.
Fluorescence microscopy nuclear protein visualization requires red-green fluorophore pairs to minimize chromatic aberration when imaging phosphorylated proteins translocating to nucleus post-LPS stimulation.
Chromatic aberration increases with shorter wavelengths: blue (405 nm) > green (488 nm) > red (594 nm). Nuclear imaging through 170 μm glass + cytoplasm demands longest practical wavelengths.
Fluorophore Performance Table
| Combination | Chromatic Shift | Nuclear Clarity | GATE Choice |
| Red+Green | Low (~20 nm) | Excellent | A, B, C |
| Blue+Green | High (~50-80 nm) | Poor | D Wrong |
| Red+Blue | High | Poor | N/A |
Optimal pairs: Alexa Fluor 488 (green) + 594 (red); avoids DAPI channel entirely for nuclear work.
Protocol for LPS Stimulation
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Stimulate cells with LPS → X/Y phosphorylation → nuclear import
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Fix PFA → anti-pX-FITC + anti-pY-Cy3
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Image 488/594 excitation → minimal aberration → clear co-localization
Exam Key: Nuclear = Red+Green. Blue = plasma membrane/DAPI only. Chromatic aberration disqualifies D.
