Diana E. Bedolla

Soft X-Ray Microscopy Radiation Damage On Fixed Cells Investigated With Synchrotron Radiation FTIR Microscopy

Radiation damage of biological samples remains a limiting factor in high resolution X-ray microscopy (XRM). Several studies have attempted to evaluate the extent and the effects of radiation damage, proposing strategies to minimise or prevent it. The present work aims to assess the impact of soft X-rays on formalin fixed cells on a systematic manner. The novelty of this approach resides on investigating the radiation damage not only with XRM, as often reported in relevant literature on the topic, but by coupling it with two additional independent non-destructive microscopy methods: Atomic Force Microscopy (AFM) and FTIR Microscopy (FTIRM). Human Embryonic Kidney 293 cells were exposed to different radiation doses at 1u2009keV. In order to reveal possible morphological and biochemical changes, the irradiated cells were systematically analysed with AFM and FTIRM before and after. Results reveal that while cell morphology is not substantially affected, cellular biochemical profile changes significantly and progressively when increasing dose, resulting in a severe breakdown of the covalent bonding network. This information impacts most soft XRM studies on fixed cells and adds an in-depth understanding of the radiation damage for developing better prevention strategies.

Time-Resolved FT-IR Microspectroscopy of Protein Aggregation Induced by Heat-Shock in Live Cells

Maintaining the correct folding of cellular proteins is essential for preserving cellular homeostasis. Protein dishomeostasis, aberrant protein folding, and protein aggregation are indeed involved in several diseases including cancer, aging-associated, and neurodegenerative disorders. Accumulation of protein aggregates can also be induced from a variety of stressful conditions, such as temperature increase or oxidative stress. In this work, we monitored by Fourier transform-infrared (FT-IR) microspectroscopy the response of live breast cancer MCF-7 and mammary breast adenocarcinoma MDA-MB 231 cell lines to severe heat-shock (HS), caused by the rise of the cellular medium temperature from 37 ± 0.5 °C to 42 ± 0.5 °C. Through the study of the time-evolution of the second derivatives of the spectra and by the 2D correlation analysis of FT-IR absorbance data, we were able to identify a common sudden heat-shock response (HSR) among the two cell lines. The hyperfluidization of mammalian cell membranes, the transient increment of the signal lipids, as well as the alteration of proteome profile were all monitored within the first 40 min of stress application, while the persistent intracellular accumulation of extended β-folded protein aggregates was detected after 40 min up to 2 h. As a whole, this paper offers a further prove of the diagnostic capabilities of FT-IR microspectroscopy for monitoring in real-time the biochemical rearrangements undergone by live cells upon external stimulation.

Apoptotic pathways of U937 leukemic monocytes investigated by infrared microspectroscopy and flow cytometry

Apoptosis is a strictly regulated cell death mechanism that plays a pivotal role in the normal evolution of multicellular organisms. Its misregulation has been associated with many diseases, making its early and reliable detection a key point for modern cellular biology. In this paper, we propose the use of infrared microspectroscopy (IRMS) as a label-free methodology for the detection of apoptotic-related biochemical processes induced on U937 leukemic monocytes by serum starvation and CCCP-exposure. The spectroscopic results are in agreement with parallel Flow Cytometry (FC) experiments, where plasma membrane integrity and mitochondrial activity were assessed. Spectroscopic outcomes complement FC data and allow drawing a more complete picture of the apoptotic pathways. In particular, we established that the two apoptosis-inducing treatments, cell starvation and CCCP exposure, affect the cell cycle in a different way. With the former, cell death is preceded by a cell cycle arrest, whereas the latter causes an increased cell cycle progression. Spectral data demonstrate that for both conditions apoptosis proceeds through the accumulation of lipid droplets within cells. Moreover, we were able to establish a spectral marker for DNA condensation/fragmentation: the enhancement of the PhI band component centred at ~1206 cm(-1), which is more sensitive than the relative intensity of the PhII band to which phospholipids and carbohydrates also contribute significantly. In conclusion, we demonstrate that the intrinsic multi-parametric nature of IRMS and its application on cells under physiological conditions can be well exploited for the investigation of apoptotic pathways.

Combining multiple imaging techniques at the TwinMic X-ray microscopy beamline

In synchrotron facilities, imaging techniques are on high demand from the scientific community. Those related to X-ray microscopy are among the most prominent ones. Such techniques include scanning transmission x-ray microscopy (STXM), full-field transmission x-ray microscopy (TXM), and coherent diffraction imaging (CDI) which have a wide spectrum of applications ranging from clinical and biomedical sciences to nanotechnology and cultural heritage. Their advancement is achieved through specialisation and focused studies, often requiring dedicated beamline end-stations. On the other hand, scientific applications benefit from the combination of techniques in a complementary manner. Beamlines suitably designed to offer multiple techniques, instead of a single one, can host efficiently such combinatorial studies. In this paper, we present the diverse Soft X-ray microscopy techniques in use at the TwinMic beamline at Elettra Sincrotrone Trieste, namely, STXM combined with XRF spectroscopy, full-field TXM and P...

Contribution of Ribonucleic Acid (RNA) to the Fourier Transform Infrared (FTIR) Spectrum of Eukaryotic Cells

We report on an optimized protocol for the digestion of cellular RNA, which minimally affects the cell membrane integrity, maintaining substantially unaltered the vibrational contributions of the other cellular macromolecules. The design of this protocol allowed us to collect the first Fourier transform infrared (FTIR) spectra of intact hydrated B16 mouse melanoma cells deprived of RNA and to highlight the in-cell diagnostic spectral features of it. Complementing the cellular results with the FTIR analysis of extracted RNA, ds-DNA, ss-cDNA and isolated nuclei, we verified that the spectral component centered at ∼1220 cm-1 is a good qualitative and semiquantitative marker of cellular DNA, since it is minimally affected by cellular RNA removal. Conversely, the band centered at ∼1240 cm-1, conventionally attributed to RNA, is only a qualitative marker of it, since its intensity is majorly influenced by other macromolecules containing diverse phosphate groups, such as phospholipids and phosphorylated proteins. On the other hand, we proved that the spectral contribution centered at ∼1120 cm-1 is the most reliable indicator of variations in cellular RNA levels, that better correlates with cellular metabolic activity. The achievement of these results have been made possible also by the implementation of new methods for baseline correction and automated peak fitting, presented in this paper.

Fourier transform infrared microspectroscopy reveals biochemical changes associated with glioma stem cell differentiation.

According to the cancer stem cell theory malignant glioma is incurable because of the presence of the cancer stem cells - a subpopulation of cells that are resistant to therapy and cause the recurrence of a tumor after surgical resection. Several protein markers of cancer stem cell were reported but none of those is fully reliable to grade the content of stem cells in a tumor. Hereby we propose Fourier transform infrared (FTIR) microspectroscopy as an alternative, labelfree, non-damaging and fast method to identify glioma stem cells based on their own spectral characteristics. The analysis of FTIR data revealed that in NCH421k cells, a model of glioma stem cells, the relative content of lipids is higher than in their all-trans retinoic acid-differentiated counterparts. Moreover, it has been assessed that stem cells have more rigid cellular membranes and more phosphorylated proteins, whereas after differentiation glycogen level increases. The ability of FTIR to estimate the content of stem cells in a heterogeneous sample, on the base of the identified spectral markers, and to classify stem and non-stem cells into two separate populations was probed. Although it was not possible to calculate the exact percentage of each subpopulation, we could clearly see that with the increasing amount of differentiated cells in a sample, more hits occupy the PC space previously identified as a space of differentiated cells. The present study is therefore an initial step towards the development of a FTIR based protocol in clinical practice to estimate the content of stem cells in a tumor sample.

Effects of soft X-ray radiation damage on paraffin-embedded rat tissues supported on ultralene: a chemical perspective

Radiation damage is an important aspect to be considered when analysing biological samples with X-ray techniques as it can induce chemical and structural changes in the specimens. This work aims to provide new insights into the soft X-ray induced radiation damage of the complete sample, including not only the biological tissue itself but also the substrate and embedding medium, and the tissue fixation procedure. Sample preparation and handling involves an unavoidable interaction with the sample matrix and could play an important role in the radiation-damage mechanism. To understand the influence of sample preparation and handling on radiation damage, the effects of soft X-ray exposure at different doses on ultralene, paraffin and on paraffin-embedded rat tissues were studied using Fourier-transform infrared (FTIR) microspectroscopy and X-ray microscopy. Tissues were preserved with three different commonly used fixatives: formalin, glutaraldehyde and Karnovsky. FTIR results showed that ultralene and paraffin undergo a dose-dependent degradation of their vibrational profiles, consistent with radiation-induced oxidative damage. In addition, formalin fixative has been shown to improve the preservation of the secondary structure of proteins in tissues compared with both glutaraldehyde and Karnovsky fixation. However, conclusive considerations cannot be drawn on the optimal fixation protocol because of the interference introduced by both substrate and embedding medium in the spectral regions specific to tissue lipids, nucleic acids and carbohydrates. Notably, despite the detected alterations affecting the chemical architecture of the sample as a whole, composed of tissue, substrate and embedding medium, the structural morphology of the tissues at the micrometre scale is essentially preserved even at the highest exposure dose.

Light element distribution in fresh and frozen–thawed human ovarian tissues: a preliminary study

RESEARCH QUESTIONnDoes synchrotron X-ray fluorescence (XRF) provide novel chemical information for the evaluation of human ovarian tissue cryopreservation protocols?nnnDESIGNnTissues from five patients undergoing laparoscopic surgery for benign gynaecological conditions were fixed for microscopic analysis either immediately or after cryopreservation. After fixation, fresh and slowly frozen samples were selected by light microscopy and transmission electron microscopy, and subsequently analysed with synchrotron XRF microscopy at different incident energies.nnnRESULTSnThe distributions of elements detected at 7.3u2009keV (S, P, K, Cl, Fe, and Os) and 1.5u2009keV (Na and Mg) were related to the changes revealed by light microscopy and transmission electron microscopy analyses. The light elements showed highly informative findings. The S distribution was found to be an indicator of extracellular component changes in the stromal tissues of the freeze-stored samples, further revealed by the transmission electron microscopy analyses. Low-quality follicles, frequent in the freeze-thawed tissues, showed a high Na level in the ooplasm. On the contrary, good-quality follicles were detected by a homogeneous Cl distribution. The occurrence of vacuolated follicles increased after cryopreservation, and the XRF analyses showed that the vacuolar structures contained mainly Cl and Na.nnnCONCLUSIONSnThe study demonstrates that elemental imaging techniques, particularly revealing the distribution of light elements, could be useful in establishing new cryopreservation protocols.