Vertical Noise Pattern in TIRF Images

I am using a TIRF microscope coupled with an EMCCD camera to image cells. I get a wierd pattern in the vertical direction which increases with increase in signal, camerga gain, laser intensity. Can anyone help.

This is referred to as image smear, and it an expected artifact of the shutterless frame transfer method used to read the EM-CCD chip. This effect is always happening, but is not detectable when imaging low light level samples, which EM-CCDs were designed for. The only way to avoid it is to reduce the intensity of the image (eg, exposure time, gain, laser power, etc).

Thank you Dr. Jennifer… Will try out these conditions and let you know. Also do you think EMCCD might not be a good choice for TIRF because TIRF in general enhances the signal?

To be clear, TIRF doesn’t enhance the signal - the signal is still a product of the amount of fluorophore and the illumination intensity. TIRF dramatically reduces background from out of focus fluorophore, which makes low signals easier to detect.
To (sort of!) answer your question: Not necessarily. The best choice of detector depends more on the sample and experimental question rather than the imaging modality. I would recommend that you do not use the electron multiplication (the EM in EM-CCD) with bright signals, but rather use the camera in standard CCD mode. EM can be turned off in the software, and is usually called gain or multiplication. Note that there is another type of gain used in CCDs that you will also come across in the software - analog gain. You are looking for the former.
Here’s a good book chapter on cameras you may want to check out:

Thank you again… I will surely go through the suggested book chapter.