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The Soft Observer Effect

Wednesday, September 16, 2015

Lisa Vaccari, Beamlines Microscopy/Diffraction group:
Alessandra Gianoncelli, Beamline scientist:

X-ray microscopy is a well-established powerful tool for investigating complex biological systems providing new insights on the biochemical processes happening at cellular and sub-cellular level. It combines X-ray imaging capabilities, such as absorption and phase contrast, with chemical information through X-ray fluorescence and XANES spectroscopies. However, radiation damage of biological samples remains a limiting factor in high resolution X-ray microscopy, especially when operating in the soft X-ray regime. Soft X-rays intrinsically cause more damage than hard X-rays due to the high absorption cross sections of C, N and O K-shells, the main organic matter constituents.

Top panels show the chemical images obtained by FTIRM (54 × 96 μm2 at 6 μm lateral resolution) of the distribution of cellular Lipids (Area integral of the spectral region 2988–2830 cm−1 cell during the five experimental steps: Air-dried, Vacuum-dried (p <5∙10−5 mbar, 1h30), low X-ray dose exposure compatible with low resolution STXM (2·10-6 Gy), medium X-ray dose exposure compatible with high-resolution STXM (2·10-7 Gy) and finally high X-ray dose exposure compatible with XRF (6·10-8 Gy). Bottom panels show the corresponding AFM images. ) of an individual HEK293T formalin-fixed. Click for full-sized image.

The extent to which the lateral resolution can be pushed without unacceptable sample degradation is still an open question. This issue concerns the whole X-ray microscopy scientific community, and it is becoming even more urgent in recent years since we are moving towards more intense and nanometer focused X-ray beams. Several studies were performed in the past, highlighting that the nature and the extent of the radiation damage strongly depend on the sample matrix composition, sample preparation, X-ray wavelength and exposure dose. However, often the very same technique used for inducing the radiation damage has been employed for testing its effects.

A study, devoted to the investigation of radiation damage induced by soft X- ray exposure to chemically fixed cells, was performed at the TwinMic and SISSI beamlines of Elettra and recently published on NPG’s Scientific Reports. As Alessandra Gianoncelli, responsible for the TwinMic beamline, points out, “we investigated for the first time the radiation damage in a systematic way and using three different microscopy techniques. X-ray microscopy was used to expose formalin-fixed Human Embryonic Kidney 293 cells to soft X-rays (1 KeV) at different doses (up to 108 Gy), while two other independent and non-destructive microscopy techniques to probe the dose- dependent effects: SR FTIR microscopy (FTIRM) and Atomic force microscopy (AFM).”

Beside mass thickness variations, previously assessed by other authors, AFM analysis reveals that cell morphology is not substantially affected even at higher exposure doses, while indeed nanometric pits and bulges increase in number and size when increasing the exposure time. These evidences suggest the collapse of the cellular structure, a hypothesis corroborated by the covalent bonding network desegregation highlighted by IRMS. Overall, FTIRM suggests that low-energy X-rays on formalin fixed cells primarily induce the oligomerization of bio-macromolecules and then affect their constitutive monomers down to the formation of small, and possibly volatile compounds.

“By imaging by AFM and IRMS the same cells before and after each exposure we could de-couple the source of the damage from the probes of it. Similar observations could be of interest for samples coming from cultural heritage or environmental science fields.” adds Lisa Vaccari, responsible for the Life Sciences branch of SISSI beamline.

The findings allow the scientific community to get new insights on the effect of X-ray exposure on the biochemical response of the organic matrix. Ultimately they will allow the scientific community to better select the experimental conditions/methodologies for performing X-ray microscopy studies.

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