2) (4 pts) Using the fluorescence microscope shown in part A of the figure, you

Question

2) (4 pts) Using the fluorescence microscope shown in part A of the figure, you wish to view a protein within the cell. You have an antibody that recognizes your protein and a secondary antibody that is coupled to rhodamine B. filter 2 filter 1 light Your lab has the following barrier filters i. A filter that passes wavelengths between 460nm and 500nm ii. A filter that passes wavelength between 520nm and 565nm. ii. A filter that passes all wavelengths above 420nm. iv. A filter that passes all wavelengths above 520nm. v. A filter that passes wavelengths between 600nm and 640nm vi. A filter that passes all wavelengths above 660nm. Answer the following questions about how you would set up the microscope to view your protein (hint you may need to use Fig. 9-13 of your book) a. Which filter would you select to place between the light source and the sample (filter 1)? b. Which filter would you select to place between the sample and the eyepiece (filter 2)? c. For a beam-splitting mirror, which wavelengths would be reflected and which would be transmitted?

Explanation / Answer

a. The filter to be used between light source and sample (filter 1) is option ii - filter that passes light of wavelength ranging from 520 to 560 nm.

This filter is the excitation filter ( filter 1 ). Rhodamine B has a fluorescence excitation at around 540nm which falls within the range of 530-560nm. This filter allows only passage of wavelength within this range to allow proper excitation of the protein molecules in the cell.

b. Filter 2 would be option v- wavelength range from 600 to 640nm because 625nm is the wavelength of emission for Rhodamine B.

c. For a beam splitting mirror, the wavelength that is reflected is 540 nm ( excitation wavelength ) and the transmitted wavelength is the emission wavelength at or above 625nm which reaches the eye piece.

This way a beam splitting mirror allows only selected wavelength to fall on the specimen and ensures that the excitation of the Rhodamine B is not contaminated by any other wavelength, this reduces background noise of the data collected. The emission wavelength is the wavelength emitted after Rhodamine B is excited at its proper wavelength. This process is called fluorescence. The difference between excitation and emission spectra allows detection of presence of the protein. This unique property of some substances is used in fluorescence microscopy.

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