Even with as low as 0.5 microlitre volume of FM-dye, laser power as low as 10%, penetration depth of 25nm, my cell sample illuminates like it does epifluorescence. I am using Olympus CellLineTIRF 4.
Is your beam focused? The best way to do this is to 1: Position the objective to focus on the specimen. 2: Take the sample off the stage and clean the oil off the lens. 3: Turn on your laser with the beam going straight up through the lens and look at the beam spot on the ceiling directly above the lens. 4: Adjust the TIRF illuminator focusing lens so that you make the beam as tight as possible (smallest spot on the ceiling). This lens may be on the TIRF illuminator or perhaps controllable in the software. 5: Put the sample back on, fine tune objective lens focus, and then adjust the beam angle to get TIRF.
Why do this? If the beam is defocused it will be too large and spill outside of the TIR zone when it enters the back focal plane. You will not get good TIRF. Since beam focusing is dependent on the objective lens height, it works best to adjust objective lens height first so the lens is roughly at the position where you will image.
Be careful when adjusting beam focus and angle. Talk to an expert or safety official at your institution about best procedures for adjusting high intensity laser optics.
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