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. Author manuscript; available in PMC: 2019 Jan 3.
Published in final edited form as: J Vis Exp. 2014 Oct 20;(92):e51805. doi: 10.3791/51805

Figure 2: Finding the TIRFM field.

Figure 2:

(A) The plasma membrane imaged in confocal. The left panel depicts cells in confocal with a focus on the center of the cell. The plasma membrane is only seen as a “ring” around the cell. Focusing down to the basal plasma membrane, adjacent to the coverslip causes the cell to be filled in with a dim fluorescence. The plasma membrane “ring” should not be visible in the thinner TIRF field. (B) The basal plasma membrane with a shallower TIRF angle producing conventional epifluorescence illumination through the sample. This illuminates the entire cell and much out of focus fluorescence from the top of the cell is present. (C) The plasma membrane in TIRF. Focusing on filopodia helps to find this plane. Notice that it is a smooth single plane. Scale bars are 10 μm. Please click here to view a larger version of this figure.