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Updated: Jan 2, 2026

Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope
Published on: April 7, 2014
Toward the quantitative the interpretation of hole-free phase plate images in a transmission electron microscope
Ken Harada1, Marek Malac2, Misa Hayashida3
1RIKEN, Center for Emergent Matter Science (CEMS), Hatoyama, Saitama 350-0395, Japan.
Abstract:
We present progress toward the quantitative interpretation of phase contrast images obtained using a hole-free phase plate (HFPP) in a transmission electron microscope (TEM). We consider a sinusoidal phase grating test object composed of ~5 nm deep groves in a ~13 nm thick amorphous silicon membrane. The periodic grating splits the beam current into direct beam and diffracted side beams in the focal plane of the imaging lens, where the HFPP is located. The physical separation between the beams allows for a detailed study of the HFPP phase shift evolution and its effect on image contrast. The residual phase shift of the electron beam footprint on the phase plate was measured by electron holography and used as input to image simulations that were compared to experimental data. Our results confirm that phase contrast is established by the phase difference between the direct and side beams, which we can estimate by fitting the image contrast evolution in time with an analytical formula describing the image intensity of a sinusoidal strong phase object. We also observed contrast reversal and frequency doubling of the grating image with time, which we interpret as the phase contrast arising from the interference between side beams becoming dominant. Another observation is the lateral displacement of the image fringes, which can be accounted for by a phase difference between the side beams.
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