In FRAP, how do we decide the wavelength of emission filter to be used? We are presently using a LASER of 488nm, Dichroic mirror (DM) of 510nm and emission filter of 520nm. Can we use an emission filter of 505nm and how it is going to affect the counts we obtain?
Secondly, if we remove the emission filter from the path we observe some leakage of LASER light and a bluish tinge is observed along with the fluorescence signal even if DM is present. Is it essential to have emission filter in path in addition to DM?
You can use the 505nm emission filter if it is long pass filter, or if it is a band pass filter extending beyond 510nm, as this is your dichroic cut-off. How it affects the counts you obtain will depend on what the two filter specs are (the 520 and 505), and where the emission maximum of your sample lies.
Whether it is essential to have an emission filter as well as the dichroic depends on how high contrast the sample you are looking at is, but since dichroics are often only 90% efficient, it is usually a good idea to use an emission filter.
Hi Santripti, we used wavelenth of 593 nm using a WPA Lightwave S2000 spectrometer.
You can use the 505nm emission filter if it is long pass filter, or if it is a band pass filter extending beyond 510nm, as this is your dichroic cut-off. How it affects the counts you obtain will depend on what the two filter specs are (the 520 and 505), and where the emission maximum of your sample lies.
Whether it is essential to have an emission filter as well as the dichroic depends on how high contrast the sample you are looking at is, but since dichroics are often only 90% efficient, it is usually a good idea to use an emission filter.
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