The role of electron irradiation history in liquid cell transmission electron microscopy

Abstract

New nanofluidic LC-TEM devices enable controlling and understanding electron irradiation history effects on liquid samples.

Considerations for imaging thick, low contrast, and beam sensitive samples with liquid cell transmission electron microscopy

“…The final column in figure 3 shows the first and last image from the damage series aligned and overlaid on top of one another allowing for comparison of changes in the cells between images. Both LC-TEM damage series illustrate the same beam driven cell shrinking at equivalent onsets and similar to what has been reported previously (12,17), indicating the presence of gold in the growth media does not considerably increase the beam driven morphological changes observed in the data. Figures 3c and 3d show comparative damage series of C. metallidurans grown with (3c) and without (3d) gold, but imaged with cryo-EM rather than LC-TEM.…”

“…Because of this, ice thicknesses for cryo-EM samples were intentionally made thicker than ideal for appropriate comparison to the LC-TEM data. We have previously shown LC-TEM results on C. metallidurans that show increased detail when compared with the data shown here when a very thin sample was achieved (17), although replication of these sample thicknesses across the multiple samples imaged here was not accomplished within the experimental time frame despite numerous attempts. As a result the data here is not representative of what may be maximally achieved for resolution and SNR with LC-TEM of biological samples, but a comparison of which imaging modalities give the strongest SNR for samples at comparable thicknesses.…”

“…It was also found that cells acquired with low convergence angle BF-STEM were more commonly in focus than images acquired with high convergence angles. While patterned grid bars were used for focusing prior to image acquisition (17), the narrow depth of focus of high convergence angle STEM has increased sensitivity to variations in sample position due to membrane bulging or small sample tilts laterally from where focus was captured. Therefore, if STEM imaging is preferred over TEM imaging our results show that low convergence angles should be used due to increased SNR, contrast, and depth of focus.…”

“…However, cumulative electron fluxes in cryo-EM above 100 e -/Å 2 often begin to cause bubble formation from hydrogen bubble generation which can cause visible image distortions (28). We have previously reported beam driven shrinking of C. metallidurans in LC-TEM for cumulative electron fluxes considerably lower than those described for cryo-EM of whole cells at 1-4 e -/Å 2 (17). These cells had been cultured in media which had been supplemented with 50 µM AuCl.…”

“…The electron irradiation sensitivity of biological samples additionally necessitates low electron fluxes for image formation during LC-TEM, likely at reduced values than those established for cryo-EM (17), which further decreases SNR and contrast. As such there is a need to optimize imaging conditions such that SNR, contrast, and resolution are maximized for images collected from thick samples with low electron fluxes.…”