Whole-cell scan using automatic variable-angle and variable-illumination-depth pseudo-total internal reflection fluorescence microscopy.

An automatic calibration and angle-scanning prism-type total internal reflection fluorescence microscope (TIRFM) was modified to function in both TIRFM and pseudo-TIRFM modes. When the incident angle of the excitation laser beam was controlled to be larger than the critical angle, the instrument served as a variable-angle TIRFM. A homemade computer program automatically calibrates the laser illumination spot in the sample to overlap with the center of the microscope's field of view. Then, by measuring the fluorescence intensities at different incident angles, the z-positions of fluorescent nanospheres close to the cell basolateral membrane can be extracted. When the incident angle is reduced to be in the subcritical range, the instrument works as a pseudo-TIRFM. The whole cell body from bottom to top can be imaged in a vertical scan process. Furthermore, the illumination field depth in the pseudo-TIRFM can be controlled by changing the incident angle or the horizontal position of the laser spot.