Anal. Chem. 2007, 79, 7353-7359
Subcellular Speciation Analysis of Trace Element Oxidation States Using Synchrotron Radiation Micro-X-ray Absorption Near-Edge Structure Thomas Bacquart,† Guillaume Deve`s,† Asuncio´n Carmona,† Re´mi Tucoulou,‡ Sylvain Bohic,‡ and Richard Ortega*,†
Cellular Chemical Imaging and Speciation Group, CNAB UMR 5084, CNRS/Universite´ de Bordeaux 1, BP 120 Le Haut Vigneau, 33175 Gradignan cedex, France, and Microfluorescence, Imaging and Diffraction, ID22 Beamline, European Synchrotron Radiation Facility, 38043 Grenoble cedex, France
Identification of chemical species at a subcellular level is a key to understand the mechanisms involved in the biology of chemical elements. When performed with a microbeam, X-ray absorption near-edge structure (microXANES) enables the direct speciation analysis of oxidation states in subcellular compartments avoiding cell fractionation and other preparation steps that might modify the chemical species. Here we report the principal characteristics in terms of spatial resolution, detection limit, reproducibility, and repeatability of a micro-XANES experimental setup based on Kirkpatrick-Baez X-ray focusing optics that maintains high flux of incoming radiation (>1011 photons/s) at micrometric spatial resolution (1.5 × 4.0 µm2). Applications and limitations of this setup are illustrated by examples of iron and arsenic absorption spectra obtained from the cytosol, nucleus, and mitochondrial network of cultured cells. A better repeatability and sensitivity with no oxidation state modification and minimal beam damage is achieved when cells are analyzed in a frozen hydrated state, as compared to freeze-dried cells. This original experimental setup can now be applied for the direct speciation analysis of most trace elements at the subcellular level. Inorganic chemical elements are involved in numerous biological processes. Some inorganic elements are essential for life, some are toxic (i.e., heavy metals), and six elements are classified as carcinogens for humans (Be, Cr, Ni, As, Cd, Pb). In order to understand complex biomedical issues involving inorganic elements, the chemical species of the elements must be considered: oxidation state, coordination, and/or complex or molecular structure will affect bioavailability, distribution, and toxicity. The International Union of Pure and Applied Chemistry (IUPAC) defines “speciation analysis” as the analytical activity of identifying and/or measuring the quantities of one or more individual chemical species in a sample.1 Speciation analysis has to overcome * To whom correspondence should be addressed. Phone: +33 557 120 907. Fax: +33 557 120 900. E-mail:
[email protected]. † Universite´ de Bordeaux. ‡ European Synchrotron Radiation Facility. (1) Templeton, D. M.; Ariese, F.; Cornelis, R.; Danielsson, L. G.; Muntau, H.; van Leeuwen, H. P.; Lobinski, R. Pure Appl. Chem. 2000, 72, 1453-1470. 10.1021/ac0711135 CCC: $37.00 Published on Web 09/07/2007
© 2007 American Chemical Society
two major challenges: (1) inorganic chemical elements are usually present in trace amount in biological systems requiring highly sensitive methods of analysis, and (2) identification of the chemical species must be performed without modification of the original compound. Very few analytical techniques fulfill these criteria, as recently reviewed.2 Although direct analysis is ideally the best approach for speciation, it is often limited by a lack of sensitivity and separation power. This is why hyphenated techniques, combining a separation method coupled with a highly sensitive detection technique, have been extensively developed these late years. However, hyphenated techniques have some drawbacks with respect to possible species transformation during analysis. A further difficulty arises at the time to identify chemical species in subcellular compartments. Usually, cellular compartments are separated by cell fractionation followed by differential ultracentrifugation, but again this sample preparation is likely to alter the chemical species to be identified. The specific aim of this work was to develop, characterize, and apply to real samples a sensitive and spatially resolved method for the direct speciation analysis of inorganic elements at the subcellular level, using synchrotron radiation micro-X-ray absorption near-edge structure (XANES). XANES is a well-established method to characterize the oxidation state of chemical elements of bulk biological samples.3 In the process of absorption of X-ray photons, their energy is converted into kinetic energy and transferred to orbital electrons of absorbing atoms. The photoelectric cross section features abrupt discontinuities, called absorption edges, at photon energies corresponding to those of various electronic levels in the atom. The oxidation state of the absorbing atom can be determined from the absorption edge energy and from preedge structures. The recent development of X-ray focusing optics allows the application of XANES at the microscopic level and offers unique characteristics for direct subcellular speciation analysis. There are only few reports of direct speciation of trace elements within cellular compartments; all are based on synchrotron radiation micro-XANES.4-9 In order to improve the sensitivity of micro-XANES, we adapted an experi(2) Lobinski, R.; Moulin, C.; Ortega, R. Biochimie 2006, 88, 1591-1604. (3) Gunter, K. K.; Miller, L. M.; Aschner, M.; Eliseev, R.; Depuis, D.; Gavin, C. E.; Gunter, T. E. Neurotoxicology 2002, 23, 127-146. (4) Ortega, R.; Deve`s, G.; Bohic, S.; Simionovici, A.; Me´nez, B.; Bonnin-Mosbah, M. Nucl. Instrum. Methods Phys. Res., Sect. B 2001, B181, 480-484.
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Figure 1. Schematic view of the Kirkpatrick-Baez mirrors (K-B) based micro-XANES experimental setup. The X-ray beam produced by the synchrotron facility is focused down to 1.5 µm vertical and 4.0 µm horizontal sizes over the 6-18 keV range. A video microscope is used to localize the subcellular compartments to be analyzed. An ionization chamber measures incoming radiation intensity (I0), while a Si(Li) detector measures the emitted X-ray fluorescence (If). X-ray fluorescence is used both to image chemical elements in cells and to perform XANES.
mental setup based on synchrotron X-ray fluorescence microanalysis10 with Kirkpatrick-Baez (K-B) optics delivering a high photon flux (>1011 photons/s) to perform micro-XANES on cultured cells. This article reports the main characteristics of this experimental setup for direct speciation analysis in terms of spatial resolution, detection limit, reproducibility, and repeatability. This article also reports examples of speciation analysis of iron and arsenic in different subcellular compartments, cytosol, nucleus, and mitochondrial network of cancer cells and neurons. These examples are typical of the study of a physiological element, iron, which is present in relatively high amount in cells and of the study of a toxic and carcinogenic element, arsenic, present in lower amount. EXPERIMENTAL SECTION Micro-XANES Setup and Sample Environment. MicroXANES experiments (Figure 1) were conducted at the European Synchrotron Radiation Facility (ESRF) at ID22 beamline.11 X-rays (5) Yoshida, S.; Ide-Ektessabi, A.; Fujisawa, S. J. Synchrotron Radiat. 2001, 8, 998-1000. (6) Kemner, K. M.; Kelly, S. D.; Lai, B.; Maser, J.; O’Loughlin, E. J.; SholtoDouglas, D.; Cai, Z.; Schneegurt, M. A.; Kulpa, C. F., Jr.; Nealson, K. H. Science 2004, 306, 686-687. (7) Ortega, R.; Fayard, B.; Salome´, M.; Deve`s, G.; Susini, J. Chem. Res. Toxicol. 2005, 18, 1512-1519. (8) Harris, H. H.; Levina, A.; Dillon, C. T.; Mulyani, I.; Lai, B.; Cai, Z.; Lay, P. A. J. Biol. Inorg. Chem. 2005, 10, 105-118. (9) Yang, L.; McRae, R.; Henary, M. M.; Patel, R.; Lai, B.; Vogt, S.; Fahrni, C. J. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 11179-11184. (10) Ortega, R.; Bohic, S.; Tucoulou, R.; Somogyi, A.; Deve`s, G. Anal. Chem. 2004, 76, 309-314.
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are produced by a high-β undulator. The source is 800 µm × 30 µm (full width at half-maximum (fwhm)) and situated at a distance of 42 m away from the focusing optics. The X-ray energy was defined using a Khozu fixed-exit double monochromator with an energy resolution of approximately 1.0 eV. The micro-fluorescence setup was mounted behind a K-B focusing system,10 producing a beam spot size of 1.5 × 4 µm2 with a high photon flux of 1.5 × 1011 photons/s at 11.9 keV. The K-B system designed and fabricated at the ESRF is achromatic over the 6-18 keV range thus providing a fixed and stable microbeam position when energy is scanned,12 which is a prerequisite for subcellular XANES analysis. For diluted samples analysis, XANES experiments are performed in fluorescence mode to optimize detection rates and improve sensitivity. In this mode, the absorption coefficient µ(E) is proportional to the fluorescence intensity (If) normalized by the incident beam intensity (I0). The I0 was measured using an Ar-filled ionization chamber placed between the K-B mirrors and the sample. The If was recorded with a Si(Li) energy dispersive fluorescence detector, of 30 mm2 crystal active surface, placed at 90° from the incoming beam axis. The detector nose was equipped with a 1 mm i.d. Ta collimator reducing the contribution of photons scattered in air along the incoming beam path. The sampledetector distance was set at 10 mm. The sample was mounted onto a motorized stage, tilted at 45° from the incident beam direction. A video-zoom microscope, placed perpendicular to the sample, was used to seek and align cells to be analyzed with the microbeam. The whole sample environment was placed in a dried N2 atmosphere. Experiments were performed either at room temperature, for freeze-dried cell analysis, or using a liquid nitrogen cryostream at -100 °C, allowing sample to be kept frozen over several hours during the experiment. XANES analysis at the iron absorption K edge (7.112 keV) was performed in fluorescence mode by scanning the preedge (7.0377.082 keV), at a rate of 5 s/step and with a 1.0 eV energy step, then the edge (7.082-7.142 keV) at a rate of 5 s/step and with 0.5 eV energy step, and the postedge (7.142-7.200 keV) at a rate of 5 s/step and with a 1.0 eV energy step. The energy calibration was obtained using the first inflection point of the Fe(0) K edge at 7.112 keV. XANES analysis at the arsenic absorption K edge (11.867 keV) was performed in fluorescence mode scanning the 11.840-11.900 keV energy range, at a rate of 3 s/step and with a 0.5 eV energy step. The energy calibration was obtained with a gold foil using the first inflection point of the Au L3 absorption edge at 11.919 keV. In practice, energy scans are repeated several times for each position of interest, from 2 to 10 times according to the analyte concentration, and XANES spectra are summed in order to improve spectral statistics. Although the random noise is relatively more important for such mode of acquisition using short acquisition times as compared to a single acquisition mode with longer times, it enables to monitor beam damage by comparing consecutive X-ray absorption spectra (see Radiation Damage section).13 (11) Somogyi, A.; Tucoulou, R.; Martinez-Criado, G.; Homs, A.; Cauzid, J.; Bleuet, P.; Bohic, S.; Simionovici, A. J. Synchrotron Radiat. 2005, 12, 208-215. (12) Hignette, O.; Rostaing, G.; Cloetens, P.; Ludwig, W.; Freund, A. K. Proc. SPIEsInt. Soc. Opt. Eng. 2001, 4499, 105-116. (13) Holton, J. M. J. Synchrotron Radiat. 2007, 14, 51-72.
Data Treatment. Data treatment was performed using XOP14 and XANDA15 software. The XOP program is used to divide X-ray fluorescence intensity by incoming intensity (If/I0), determine online the first derivative, and sum the spectra obtained successively of the same region before normalization. The XANDA program is designed for the analysis of XANES data and is used for preedge background subtraction, normalization of absorption to 1 after the absorption edge, and to determine the maximum of the absorption spectrum (white line) and first-derivative energies. The white line and first-derivative energies can both be used to discriminate between oxidation states.3 Results are expressed using the white line energy in the case of arsenic, and the firstderivative position in the case of iron, in order to directly compare our results with the data published in the literature. The subcellular oxidation states were identified by comparison with reference compounds: Fe(0), Fe(II) sulfate, and Fe(III) nitrate for iron, and As(II) sulfide, As(III) oxide, As(V) oxide, and dimethylarsinic acid (DMA(V)) for arsenic. Cell Culture and Sample Preparation. PC12 rat neuronal cells16 and IGROV1 human ovarian adenocarcinoma cells17 were cultured onto microanalysis sample holders as adapted from the description reported in a previous article.18 Briefly, cells were grown onto 2 µm thick polycarbonate film treated with 2% gelatin gel in culture medium supplemented with fetal bovine serum. Cells were incubated at 37.5 °C in CO2 humidity saturated atmosphere. PC12 cells were exposed during 24 h to a subcytotoxic concentration (50 µM) of FeSO4, which is directly soluble in culture medium at this concentration. IGROV1 cells were exposed to arsenic trioxide at IC50 concentration (25 µM) during 24 h. As2O3 is not directly soluble in culture medium. It is completely dissolved by adding NaOH to a stock solution in ultrapure water. A few microliters of arsenic stock solution are then diluted to the suited concentration in culture medium, and the pH is adjusted with HCl from an initial value of about 9 to 7.4. Iron and arsenic solutions in culture medium are filtered on a 0.2 µm membrane for sterilization. After metal exposure, cells were extensively washed with a physiological buffer to remove remaining traces of iron, or arsenic, bound aspecifically to the cells. Then cells were either cryofixed into liquid nitrogen chilled isopentane at -160 °C and freeze-dried into a cryostat at -35 °C to be analyzed at room temperature or cryofixed and directly stored in liquid nitrogen until analysis in their frozen hydrated state at -100 °C (Figure 2). Cells were observed by light microscopy (Olympus BX51) before cryofixation, and pictures were obtained on selected cells to identify the nucleus and cytosol areas. Localization of mitochondria was done using a specific marker for mitochondrial membrane, rhodamine123. Powdered rhodamine123 was dissolved into dimethyl sulfoxide and then diluted to 10 µM in nutritive RPMI 1640 medium without phenol red. Cells were then incubated in this medium for 1 h at 37 °C and in 5% CO2 atmosphere. For (14) Sa´nchez del Rı´o, M.; Dejus, R. J. Proc. SPIEsInt. Soc. Opt. Eng. 1998, 3448, 340-345. (15) Klementiev, K. V. XANES dactyloscope for Windows; freeware, available at www.desy.de/∼klmn/xanda.html. (16) Greene, L. A.; Tischler, A. S. Proc. Natl. Acad. Sci. U.S.A. 1976, 73, 24242428. (17) Benard, J.; Da Silva, J.; De Blois, M. C.; Boyer, P.; Duvillard, P.; Chiric, E.; Riou, G. Cancer Res. 1985, 45, 4970-4979. (18) Ortega, R.; Moretto, P.; Fajac, A., Be´nard, J.; Llabador, Y.; Simonoff, M. Cell. Mol. Biol. 1996, 42, 77-88.
Figure 2. Two analytical procedures are compared for trace element speciation in cells: at room temperature, and at low temperature. Cell culture, fluorescent marking, and cryofixation are common steps for the two sample preparation protocols. When speciation analysis is performed at room temperature a freeze-drying protocol is used after cryofixation. When speciation analysis is performed at low temperature, cells are maintained frozen hydrated after cryofixation and analyzed at -100 °C using a liquid nitrogen cryostream.
analysis of frozen hydrated cells, the fluorescent mitochondria and subcellular compartments to be analyzed were spotted in living cells using a confocal microscope (Leica DMR TCS SP2 AOBS with Ar-laser 496 nm). Safety Considerations. Inorganic arsenic is classified as a human carcinogen. The following chemicals are hazardous and should be handled carefully: arsenic(III) oxide, arsenic(II) sulfide, arsenic(V) oxide, dimethylarsinic acid. Universal precautions for the handling of chemicals and biofluids were applied. RESULTS AND DISCUSSION The micro-XANES technique was used to perform the direct speciation of arsenic and iron oxidation states on single cells. The aim of the following sections is to describe the characteristics of the micro-XANES setup at ID22 beamline in terms of spatial resolution, speciation analysis limit, reproducibility, and repeatability for single subcellular analysis. Then the scope of application and the limits of the technique, such as biological sample’s damage, are discussed based on two typical examples of analysis. The first example, the determination of iron oxidation state in neuronal cells, illustrates the study of a trace element present in relatively high amount in cells. The mean iron concentration in PC12 neuronal cells was measured by particle-induced X-ray emission (PIXE) analysis and was estimated at 75 µg/g. The second example, the speciation analysis of arsenic oxidation state in cancer cells exposed to the antitumor agent arsenic trioxide, is representative of a more diluted analyte. Arsenic mean concentration in IGROV1 cells was about 25 µg/g as measured by PIXE analysis. In both cases, PIXE analyses were performed after Analytical Chemistry, Vol. 79, No. 19, October 1, 2007
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Figure 3. Micro-XANES spectra (a.u., arbitrary units) at the arsenic absorption K edge recorded consecutively on a cell nucleus (A) at room temperature on a freeze-dried cell and (B) at -100 °C on a frozen hydrated cell. At room temperature the fluorescence intensity decreases dramatically during the third consecutive scan (analysis time, 18 min), while at low temperature the fluorescence intensity remains high after six consecutive scans (analysis time, 36 min).
micro-XANES on the same samples but on different cells. Large areas of the cellular monolayer were analyzed involving about 200 cells. Spatial Resolution and Detection Limit. The spatial resolution of the X-ray microprobe is defined by the size of the focused beam. A beam of 1.5 × 4 µm2 (vertical × horizontal) was achieved at ID22 beamline using a K-B focusing system while maintaining a high flux of 1.5 1011 photons/s at 11.9 keV. The beam size was measured using the knife edge procedure as already described.10 This beam size is well suited to analyze large intracellular compartments such as the cytosol, nucleus, or mitochondrial network in cells. The stability of the microbeam position has been verified. The beam moves of about 1 µm/1000 eV. It corresponds to a 0.1 µm movement for a XANES scan of 100 eV, which is 7356
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relatively small compared to the beam size. The limit of detection (LOD) as defined by Currie19 was evaluated from the standard deviation (s) of eight blank measures; LOD ) 3.29 s ) 4.3 µg/g. The method detection limit (MDL)20 was evaluated from microXANES analysis of arsenic-treated cells for which the arsenic concentration, 25 µg/g, is close to the expected LOD. A standard deviation of 2.58 was obtained for the maximum absorption edge intensity of seven micro-XANES analyses performed on cytosol regions, the cellular compartment presenting the lowest concentration of arsenic. For seven analyses, with six degrees of freedom and for a 99% confidence level, the t value of the Student t test is 3.14. The MDL is defined as the standard deviation multiplied by the t value; MDL ) 8.1 µg/g. The average analyzed volume being around 60 µm3 (beam size, 1.5 × 4 µm2, multiplied by cell thickness, 10 µm), this corresponds to the detection of 5 × 10-16 g of As in each analytical pixel (1.5 × 4 µm2). The MDL depends on multiple factors such as the atomic number of the analyte, the energy of the incoming beam, the integration time, the detector characteristics, etc. Therefore, the value reported here applies specifically to the speciation of arsenic oxidation state in subcellular compartments, using the described micro-XANES setup. Similarly to the concept of “limit of quantification” used for quantitative analytical methods,20 a “limit of speciation” (LOS) corresponding to 10 times the standard deviation of blank measures can be evaluated; LOS ) 13 µg/g. This value is helpful to estimate the feasibility of further experiments. Similar detection levels are expected for elements of atomic number between 20 and 40, because the cross section for X-ray fluorescence and the detection efficiency do not vary much for these elements. Reproducibility of Micro-XANES. The reproducibility of an analytical method is defined by the IUPAC such as “the closeness of agreement between independent results obtained with the same method on identical test material but under different conditions (different operators, different apparatus, different laboratories and/ or after different intervals of time)”.21 When micro-XANES measurements are compared for different reference compounds, either at Fe or As absorption K edges, an excellent reproducibility in K edge energy position is found. For example, As2O3 K edge maximum absorption energy has been reported to be 11 871.5 ( 0.5 eV by five different experiments using macro-XANES.22-26 In our experiment we obtained, after energy normalization, an As K edge of 11 871.4 ( 0.2 eV for As2O3. The same is true for Fe(II) and Fe(III) reference compounds. We measured Fe(II) and Fe(III) K edge first-derivative energies at, respectively, 7120.0 ( 0.5 eV and 7126.3 ( 0.3 eV, which are close to the values reported in (19) Currie, L. A. Anal. Chem. 1968, 40, 586-593. (20) APHA, American Water Works Association, Water Environment Federation. Standard Methods for the Examination of Water and Wastewater, 19th ed.; American Public Health Association: Washington, DC, 1995. (21) IUPAC Compendium of Chemical Terminology [Online]; International Union of Pure and Applied Chemistry. http://goldbook.iupac.org/R05305.html. (22) Wang, H. C.; Paul Wang, H.; Peng, C. Y.; Liu, S. H.; Yang, Y. W. J. Synchrotron Radiat. 2001, 8, 961. (23) Bostick, B. C.; Fendorf, S.; Manning, B. A. Geochim. Cosmochim. Acta 2003, 67, 895. (24) Deschamps, E.; Ciminelli, V. S. T.; Weidler, P. G.; Ramos, A. Y. Clays Clay Miner. 2003, 51, 197. (25) Manning, B. A.; Fendorf, S.; Suarez, D. L. Biogeochem. Environ. Important Trace Elem. 2003, 835, 57. (26) Smith, P. G.; Koch, I.; Gordon, R. A.; Mandoli, D. F.; Chapman, B. D.; Reimer, K. J. Environ. Sci. Technol. 2005, 39, 248-254.
Figure 4. (A) XANES spectra of iron(II) reference compound (Fe(II)), the cytosol (Dried-C) and the nucleus (Dried-N) of a freeze-dried cell analyzed at room temperature, the cytosol (Cryo-C) and nucleus (Cryo-N) of a frozen hydrated cell analyzed at -100 °C, and of iron(III) reference compound (Fe(III)). The dotted lines represent iron(II) and iron(III) first-derivative edge positions, respectively, at 7122.9 and 7128.7 eV. (B) Bright-field microscopy of a freeze-dried cell as observed prior to micro-XANES experiment. The cellular compartments are easily identified; analyzed zones are indicated by an arrow (C, cytosol; N, nucleus). The corresponding spectra are presented in panel A.
the literature for micro-XANES: 7120.9 ( 1.0 eV for Fe(II) and 7126.7 ( 1.0 eV for Fe(III).27,28 Repeatability and Sample’s Preservation. The repeatability of an analytical method is defined by the IUPAC such as “the closeness of agreement between independent results obtained with the same method on identical test material, under the same conditions (same operator, same apparatus, same laboratory and after short intervals of time)”.21 In the case of micro-XANES applied to single cells, repeatability is closely related to the sample’s preparation methods and the use, or not, of a cryoenvironment during analysis. Proper preparation and storage of samples are two major issues in order to avoid alteration of cell structure, chemical element redistribution, and/or modification of chemical species. We compared two different protocols: (1) cryofixation followed by freeze drying and sample storage at room temperature, that allows a good preservation of cell structure and chemical element distribution,17 and (2) cryofixation followed by direct sample storage at low temperature (liquid nitrogen), that keeps cells in frozen hydrated state. The main limitation of this second protocol is that it requires maintaining the sample frozen at all steps, including during analysis. Its main advantage is that the analysis of frozen hydrated samples at low temperature can limit thermal effects and preserves sample integrity during analysis.26,29 Both protocols were performed on the same cell lines and compared (Figure 2). To achieve better statistics, several XANES spectra obtained on the same area are usually performed and summed (see Experimental Section). At room temperature, the intensity of the fluorescence signal was maximal only for the first two microXANES spectra at the arsenic K edge (Figure 3A, first and second (27) Chwiej, J.; Adamek, D.; Szczerbowska-Boruchowska, M.; Krygowska-Wajs, A.; Wojcik, S.; Falkenberg, G.; Manka, A.; Lankosz, M. J. Biol. Inorg. Chem. 2007, 2, 204-211. (28) Ide-Ektessabi, A.; Fujisawa, S.; Yoshida, S. J. Appl. Phys. 2002, 91, 16131617. (29) Parsons, J. G.; Aldrich, M. V.; Gardea-Torresdey, J. L. Appl. Spectrosc. Rev. 2002, 37, 187-222.
spectrum). When more than two energy scans were performed at room temperature the X-ray fluorescence signal declines rapidly due to sample’s damage during irradiation (Figure 3A, thirdfifth spectra). In our experimental conditions, a single energy scan around the arsenic absorption edge lasted 6 min. It is therefore estimated that irradiation times over 18 min would irreversibly damage freeze-dried samples analyzed at room temperature (Figure 3A). When cells were analyzed at -100 °C, several energy scans could be performed without modification of the X-ray fluorescence signal intensity (Figure 3B). Up to six energy scans, corresponding to 36 min of irradiation on the same sample’s region, were performed without damaging the cell (Figure 3B). The spectra are identical in terms of shape and intensity and clearly show that no modification of sample’s integrity occurs when the sample is maintained at -100 °C. The use of cryotechniques is mandatory to perform micro-XANES during long irradiation times such as are required when the analyte concentration is close to the method speciation limit, i.e., arsenic in cancer cells. However, the analysis of freeze-dried cells at room temperature can be performed when irradiation times are shorter. This is, for example, the case for iron micro-XANES in neuronal cells where, in this case, the analyte concentration is 5 times greater than the method speciation limit and the irradiation time about 18 min (Figure 4A). In addition, it is important to note that Fe absorption spectra obtained on freeze-dried cells at room temperature, or on frozen hydrated cells analyzed at -100 °C, are similar (Figure 4A). This result indicates that the freeze-drying protocol does not modify Fe chemical state in cells. Radiation Damage. As with other techniques using ionizing radiation, radiation damage limits the study of biological specimens with micro-XANES. Our micro-XANES experiments typically involved doses ranging from 109 to 1010 Gy. These doses must be compared to the dose level of 106 to 107 Gy which is known to produce mass loss on biological samples at room temperature.30 (30) Williams, S.; Zhang, X.; Jacobsen, C.; Kirz, J.; Lindaas, S.; van’t Hof, J.; Lamm, S. S. J. Microsc. 1993, 170, 155-165.
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Indeed we observed a dramatic element loss during micro-XANES experiments at room temperature as monitored by the decrease of the white line peak intensity (Figure 3A). The damage formation can be significantly mitigated by lowering the sample temperature, primarily through the immobilization of radiolytic products in the ice matrix. For example, cryomicroscopy experiments on biological samples using soft X-rays at 113 K have shown essentially no observable mass loss with radiation doses up to 1010 Gy,31,32 corresponding to a 104 increase in radiation damage stability compared to room-temperature experiments. When micro-XANES experiments were performed at -100 °C we observed that samples were stable during irradiation, with an element loss of less than 10% after six consecutive scans as monitored with the white line height (Figure 3B). It is also known that X-ray irradiation can lead to photoreduction processes of redox metals at doses
