Off-axis electron holography for biological studies: towards the measurement of biological samples electrostatic potential

February 9, 2025

Off-axis electron holography was used to study unstained bacteriophages. We demonstrated an improvement in contrast and signal-to-noise ratio compared to bright field TEM images and showed that phase images provide information on charge variations due to the neutralization of DNA contained in these objects, opening the way to studies of the electrostatic potential of biological objects at the nanoscale.

The methodological and instrumental advances in off-axis electron holography carried out at CEMES allow achieving, for holograms obtained in vacuum, a phase sensitivity of approximately 2 mrad at a resolution of 2 nm with a typical dose of several thousand e-/Å2. These performances make holography potentially efficient for the study of biological samples that, however, do not support such high doses (only a few tens e-/Å2).

As part of an interdisciplinary thesis co-financed by the Occitanie Region and the University of Toulouse and co-directed with a team from the Centre de Biologie Intégrative (CBI) in Toulouse, and within an interdisciplinary project supported by the Miti of the CNRS, C EMES researchers evaluated the performances of holography for the structural study of biological samples studied at room temperature and in cryogenic conditions.

Our results obtained at low doses on unstained T4 and T5 bacteriophages show a significant improvement in the contrast of phase images at room temperature compared to defocused bright-field TEM images. In cryogenic studies carried out on samples embedded in vitreous ice, the phase noise resulting from the necessity to work at low doses adds to the decrease in signal/noise due to the overlapping of ice zones and leads to reduced sensitivities.

However, the researchers have also shown that holography provides access to information on local charge variations linked to variations in structural configurations inaccessible by other techniques, opening new possibilities for studying the electrostatic potential of biological objects at the nanometric scale.

Left: Cryo-TEM images of a T4 bacteriophage
Top right: Phase images of non-treated (top left and middle), and spermine-treated (top right) unstained T5 bacteriophage. Below: Phase profile of non-treated and spermine-treated unstained T5 bacteriophage head.

Contact:
Etienne Snoeck | etienne.snoeck[at]cemes.fr

Publication:
Off-axis electron holography of unstained bacteriophages: toward electrostatic potential measurement of biological samples
E. Karim, C. Gatel, A. Leforestier, S. Balor, V. Soldan, C. Plisson-Chastang, P.-E. Gleizes, and E. Snoeck
Journal of Structural Biology, Volume 217, Issue 1, 2025, 108169
DOI: https://doi.org/10.1016/j.jsb.2025.108169