A fluorescent microscope, which is shown in the figure above, uses two filters a

Question

A fluorescent microscope, which is shown in the figure above, uses two filters and a dichromatic mirror to excite the sample and capture the emitted fluorescent light. Imagine you wish to study two proteins (protein A and protein B). You tag protein A with YFP and protein B with CFP.

Assume, you have the following filters available commercially,

a. A filter that passes wavelength between 530 nm and 575 nm

b. A filter that passes wavelength above 550 nm

c. A filter that passes wavelength below 550 nm

d. A filter that passes wavelength between 450nm and 500 nm

e. A filter that passes wavelength below 400 nm

f. A filter that passes wavelength below 450 nm

g. A filter that passes wavelength between 500nm and 530 nm

h. A filter that passes wavelength between 400nm and 450 nm

1. Which ones will you select to place between the light source and the sample while performing FRET?

2. Which ones will you select to place between the sample and the eyepiece if your goal is to determine if protein A and protein B are localized within 10nm?

Explanation / Answer

1. filter to place between the light source and the sample while performing FRET:
For CFP:
h. A filter that passes wavelength between 400nm and 450 nm
For YFP:
g. A filter that passes wavelength between 500nm and 530 nm

Explanation: our goal here is to provide wavelength restricted to absorption area as we have to radiate the sampe so we use above filters. We don't use filter with broad range of wavelengths as the radiate unwanted background.

2. Which ones will you select to place between the sample and the eyepiece if your goal is to determine if protein A and protein B are localized within 10nm?

This part of question is with insufficient information as there 10nm wavelength nowhere mentioned in the graph.
However to view proteins through eyepiece, we can use following filters
For CPF:
d. A filter that passes wavelength between 450nm and 500 nm
For YPF:
a. A filter that passes wavelength between 530 nm and 575 nm

Explanation: our goal here is to provide wavelength restricted to emission as we have to capture the emitted radiation so we use above filters.

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