In-line and Off-axis Electron Holography for the Study of Biological Specimens

3D structures of biological specimens have been studied for a long time using three main techniques: NMR, X-Ray crystallography, and Cryo-electron microscopy (cryo-EM). While NMR is very challenging for proteins bigger than 50 kDa and X-Ray crystallography is exclusive to crystallizable proteins, cryo-EM has recently emerged as another powerful technique for high-resolution structural biology. Electron microscopy has however a major drawback due to the low amplitude contrast resulting from the weak electron interaction with the light elements composing biological samples. Biologists have developed methods to get around this issue, in particular the use of contrasting solutions containing heavy metals at room temperature or various types of phase plates in cryogenic conditions to enhance the TEM contrast. Herein, we analyze the performances of two electron holography techniques mostly developed for phase retrieval in material science, the focal series in-line holography and the off-axis holography techniques, to study unstained bacteriophages at room and at cryogenic temperatures. We showed that in-line holography in cryogenic conditions has great potential for imaging unstained biological specimens. In addition, thanks both to recent developments carried out at CEMES [1,2] and to the direct electron detectors performances, we show that off-axis holography enables us to retrieve the phase shift information that the electron undergoes when interacting with the low-Z biological samples. We show that these methods provide high SNR and high contrast phase images on unstained samples. (a) Focal series in-line electron holography workflow. (b) Raw defocused micrograph of the sample. Δf = -9µm. (c) In-focus amplitude contrast image extracted from the focal serie. (d) In-focus phase contrast image extracted from the focal serie. (a) Ray diagrams of the single (left) and double (right) off-axis electron holography setups. The light blue part is the reference beam that that goes through the vacuum and the dark blue part is the object beam (b) Raw off-axis hologram and a zoomed image (top right corner) showing the interference pattern between the reference beam and the object beam. (c) In-focus phase contrast image extracted from the original hologram in (b).