Exercise 5.3 A student wants to perform two-color fluorescence imaging of two di
ID: 216806 • Letter: E
Question
Exercise 5.3 A student wants to perform two-color fluorescence imaging of two different targets of nucleus and microtubules, which are stained with Alexa Fluor 555 and Alexa Fluor 594, respectively. The excitation and emission spectra of the fluorophores are shown below. The emission filters for Alexa Fluor 555 and Alexa Fluor 594 are 580/20 and 620/20, respectively with a dichroic mirror of LP570. The filters and dichroic mirror are purchased from a well-established company without any technical defects. When 561nm laser is used as the light source, both nucleus and cytoplasm channels exhibit significant fluorescent signals (a.k.a. cross-talk). Please discuss the potential cause of this cross-talk. Alexa ?or 5551a Fuor 594 100 excitation emission Laser (561 nm) ? 50 300 400 500 600 Wavelength (nm 700 800 900Explanation / Answer
The dye alexa-555 has emission spectra overlapping with the excitation spectra of the dye alexa-594. This is tha main cause of this cross-talk. When 561 nm wavelength is used for excitation, the first dye alexa-555 gets excitation and emits at slightly higher wavelength. This emission wavelength is again absorbed by dye alexa-594 and get excitation giving fluorescence from both the dyes at athe same time.