The pitfalls of in vivo imaging techniques: evidence for cellular damage caused by synchrotron X‐ray computed micro‐tomography

Abstract

Synchrotron X-ray computed micro-tomography (microCT) has emerged as a promising noninvasive technique for in vivo monitoring of xylem function, including embolism build-up under drought and hydraulic recovery following re-irrigation. Yet, the possible harmful effects of ionizing radiation on plant tissues have never been quantified. We specifically investigated the eventual damage suffered by stem living cells of three different species exposed to repeated microCT scans. Stem samples exposed to one, two or three scans were used to measure cell membrane and RNA integrity, and compared to controls never exposed to X-rays. Samples exposed to microCT scans suffered serious alterations to cell membranes, as revealed by marked increase in relative electrolyte leakage, and also underwent severe damage to RNA integrity. The negative effects of X-rays were apparent in all species tested, but the magnitude of damage and the minimum number of scans inducing negative effects were species-specific. Our data show that multiple microCT scans lead to disruption of fundamental cellular functions and processes. Hence, microCT investigation of phenomena that depend on physiological activity of living cells may produce erroneous results and lead to incorrect conclusions.

High‐resolution computed tomography reveals dynamics of desiccation and rehydration in fern petioles of a desiccation‐tolerant fern

“…Images were timed to capture the full range of hydration, starting with desiccated plants (pre-watering) through rehydrated plants (up to 25 h post-watering). The ferns resurrected similarly regardless of how many times they were imaged, and no signs of radiation damage were observed (Petruzzellis et al, 2018). In several plants, we conducted repeat scans to confirm expansion of the outer stele early in the resurrection process, since there was variation in the size of the dry stele (Supporting Information Fig.…”

Insights from in vivo micro‐CT analysis: testing the hydraulic vulnerability segmentation in Acer pseudoplatanus and Fagus sylvatica seedlings

“…1), which were fixed to the holder with Terostat putty (Teroson, Heidelberg, Germany). Thanks to the use of this sample holder, we could avoid long exposition to irradiation during the initial sample alignment, thus minimizing eventual X-ray induced cellular damage Petruzzellis et al, 2018). Overall, sample preparation, initial alignment and scan time (90 s) were performed within 10-15 min.…”

“…Choat et al, 2010;Cochard et al, 2015;Jansen et al, 2015;Brodribb et al, 2016). X-ray phase contrast micro-tomography (micro-CT) is so far the most promising method, as it is nondestructive (but see Petruzzellis et al, 2018) and allows in vivo observations of conduits status (in terms of water-vs air-filled conduits) and thus to analyse hydraulic integrity and embolism patterns within organs (e.g. Brodersen et al, 2013;Choat et al, 2015).…”

Hydraulics in the 21st century

“…The occurrence of refilling has been detected with in vivo imaging techniques in some cases (Kaufmann et al ., ; Brodersen et al ., ) but not in others (Choat et al ., ). However, it has been argued that very local damage by X‐rays in the imaging region of a stem (< 5 mm) during repeated micro‐CT scans can damage parenchyma cells in some species (Petruzzellis et al ., ), possibly hindering the vital processes that are putatively required to refill the entire stem (Lovisolo et al ., ; Laur & Hacke, ; Secchi et al ., ). Further work will be required to confirm the majority view from CT work, that refilling in plants is not possible under tension.…”

“…The first one is the possibility to observe in vivo and in real‐time the functional status of xylem conduits during drought and recovery, without damaging living wood and bark cells. While micro‐CT might not be up to this task (Petruzzellis et al ., ), the optical method applied to leaf vasculature (Brodribb et al ., ) is a very promising and nondestructive approach, but until now it has only been seldom used to observe eventual xylem refilling (K. M. Johnson et al ., ). The second methodological advancement is related to the accurate measurement of water potential in the proximity and within the eventually refilling conduits.…”