Hello everyone. Recently, I have been optimizing a TIRF microscope that has not been used for a long time and conducting research related to smFRET. However, I have encountered the problem of a high background in the 647 channel. When this microscope is switched to the eyepiece channel, the background signal values of the CY3 and CY5 channels are approximately close, about 100 - 150. However, once it is switched to the camera channel, whether the 532 or 647 laser is turned on, a very high background (around 300) will be generated in the 647 channel. Moreover, even under wide-field illumination, the 647 channel has the same high background light. Does anyone know why this is? The hardware we use is the Photometrics Evolve-512-Delta camera and Photometrics DV2. The objective lens is Nikon Apochromat TIRF 100x Oil, and the excitation optical path is manually built by aligning the laser to the ceiling.
Do you know if the eyepiece channel and the camera channel share the same filter sets ? My guess would be a different filter in each case.
Hi Adrien,Thank you for your reply, the model of the microscope we use is Nikon Ti. When taking pictures, the optical path will switch to the camera path, and there is no light at the eyepiece. After a week of exploration, I now suspect that it is a problem with the photometrics camera. For the same sample, the signal value of the fluorescence spots captured by the photometrics camera is about 4000, while the background is about 200. However, with the andor camera, the fluorescence signal value is about 15000, and the background value is also about 200. So I suspect that it is a problem with the signal acquisition of the photometrics camera.