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APPLIED BIOLOGY

To investigate the relationship between microtubules and centrioles in fixed HeLa cells using an epifluorescence microscope, a researcher plans to con

Question

To investigate the relationship between microtubules and centrioles in fixed HeLa cells using an epifluorescence microscope, a researcher plans to conduct immunostaining using antibodies against tubulin and centrin (centriolar protein). After the incubation with the primary antibodies and wash, she/he plans to use secondary antibodies that bind to the primary antibodies. Below is a list of secondary antibodies carrying various fluorophores (dyes) available to the researcher.
A. Alexa 568
B. FITC
C. Alexa 488
D. Alexa 647
Select the correct combinations of the appropriate dyes that the researcher would typically utilize to observe co-localization in an epifluorescence microscope?

A and C

B and C

A and D

C and D

Solution

Correct option is A

Explanation-

A. Alexa 568 - Suitable for co-localization experiments.
Excitation peak: ~578 nm
Emission peak: ~603 nm
Fluorescence color: Orange-Red
Microscope filter set required: TRITC filter set
Application: Alexa 568 emits in the red/orange region, making it ideal for labeling one target protein in co-localization studies when used alongside a green-emitting dye like Alexa 488. It is photostable and bright, commonly used for multicolor fluorescence imaging.
B. FITC (Fluorescein isothiocyanate) - Not suitable together with Alexa 488 for dual labeling in this case.
Excitation peak: ~495 nm
Emission peak: ~519 nm
Fluorescence color: Green
Microscope filter set required: FITC filter
Application: FITC emits green fluorescence, similar to Alexa 488, and would overlap spectrally with Alexa 488.
If FITC and Alexa 488 were both used in the same experiment, it would be difficult to distinguish between the two signals, making co-localization analysis unreliable.
C. Alexa 488 - Suitable for co-localization experiments.
Excitation peak: ~488 nm
Emission peak: ~519 nm
Fluorescence color: Green
Microscope filter set required: FITC/GFP filter set
Application: Alexa 488 is a bright and photostable green fluorophore, widely used for immunofluorescence. It is commonly paired with Alexa 568 (red) for two-color co-localization studies because of minimal spectral overlap.
D. Alexa 647 - Not preferred here for standard two-color epifluorescence setups.
Excitation peak: ~650 nm
Emission peak: ~668 nm
Fluorescence color: Far-Red
Microscope filter set required: Cy5 filter set
Application: Alexa 647 emits in the far-red region. However, many basic epifluorescence microscopes do not have Cy5 filters or the capacity to detect far-red emission without specialized setups like confocal microscopy or special filter cubes. While Alexa 647 is great for 3-color experiments in confocal microscopy, in typical epifluorescence microscopes, people often stick with green (Alexa 488) and red (Alexa 568) combinations.