Question

Explain why fluorescence is collected at an angle other than 180˚ to the excitation light, and why an emission filter is used.

Answer 1

Fluorescence is mostly observed at a 90° angle relative to excited light. The angle is maintained 90° other than 180 in order to avoid interference of the transmitted excitation light.
There is no such monochromator that is perfect and it will transmit some stray light, i.e wavelengths other than targeted ones.An ideal monochromator would only transmit light in the specified range and While measuring at a 90° angle, only the light scattered by the sample causes stray light. This results in a better signal-to-noise ratio, and lowers the detection limit by approximately a factor 10000 when compared to the 180° geometry.

The emission filter is typically a bandpass filter that passes only the wavelengths emitted by the fluorophore and blocks all undesired light outside this band – especially the excitation light. By blocking unwanted excitation energy (including UV and IR) or sample and system autofluorescence, optical filters ensure the darkest background.