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              <p>Transmission electron microscopy, especially at cryogenic temperature, is largely used for studying biological macromolecular complexes. A main difficulty of TEM imaging of biological samples is the weak amplitude contrasts due to electron diffusion on light elements that compose biological organisms. Achieving high-resolution reconstructions implies therefore the acquisition of a huge number of TEM micrographs followed by a time-consuming image analysis. This TEM constraint could be overcome by extracting the phase shift of the electron beam having interacted with a “low contrast” sample. This can be achieved by off-axis electron holography, an electron interferometric technique used in material science, but rarely in biology due to lack of sensitivity. Here, we took advantage of recent technological advances on a dedicated 300 keV TEM to re-evaluate the performance of off-axis holography on unstained T4 and T5 bacteriophages at room temperature and in cryogenic conditions. Our results demonstrate an improvement in contrast and signal-to-noise ratio at both temperatures compared to bright field TEM images, with some limitations in spatial resolution. In addition, we show that the electron beam phase shift gives information on charge variations, paving the way to electrostatic potential studies of biological objects at the nanometer scale.</p>
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