[B._Beckhoff,_et_al.]_Handbook_of_Practical_X-Ray_(b-ok.org).pdf

725 examples of applications single cell spectroscopy

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7.2.5 Examples of Applications Single Cell Spectroscopy Using PINK Beams deeper insights into biological processes and cellular analysis require nonde- structive quantitative techniques with high elemental and chemical sensitivity and high spatial resolution, such as synchrotron induced X-ray fluorescence (SXRF). In this work [70], we performed SXRF microanalysis of single cells by imaging the intracellular distribution of trace elements and pharmaco- logical doses of the anticancer drug, 4 -iodo-4 -deoxydoxorubicin (IDX). Spa- tial distribution and concentration of trace elements in tissues are important, as they are involved in some pathological conditions and in many biological functions of living organisms like metabolism and nutrition. CRL lenses of- fer several advantages compared to other focusing systems, mainly high flux tolerance, efficiency for focusing high energy X-rays up to 60 keV, and almost no spherical aberrations. These lenses used with a “pink” (polychromatic) excitation provide a fast acquisition rate and sub-ppm limits of detection. Additionally, the CRL lenses are currently the only focusing devices capable of sustaining the high flux, high heatload of a PINK beam without noticeable damage. A human ovarian adenocarcinoma (IGROV1) cell line was used for this study. The cells, grown directly on 0.2- µ m Formvar film were incubated with complete culture medium and exponentially growing cells were exposed to 5 µ M IDX. Cell monolayers were rinsed, then cryofixed and freeze-dried at 30 C. Analyses were performed at 14 keV using either pink or monochro- matic excitation. Pink excitation is produced by direct, high intensity, medium bandwidth beams from the undulator impinging onto a flat mirror. The mul- tistrip mirror spans several full undulator harmonics and decreases the beam heatload by a horizontal deflection at a grazing angle of 2.6 mrad which gives an energy cutoff of 24 keV for the Pd strip. In order to remove the contribution of lower energy harmonics, a 2-mm Al filter was used. In the pink beam, CRLs produce a flux of about 5 × 10 10 photon s 1 µ m 2 and a beam spot of 10 µ m horizontally by 1 µ m vertically for 50 Al-lenses, focal distance 713 mm. For the same spot size, CRLs with monochromatic excitation give a flux about 10 times less. The minimal detection limits (MDL) evaluated using a NIST standard reference material (SRM 1833) yielded about 30 ppb for elements
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Methodological Developments and Applications 489 such as Zn. Data analysis was performed using the WinAxil software [71] in order to correct for X-ray photon background and fit elemental X-ray lines detected in the sample. In the case of a cell monolayer, the thin sample approximation can be applied for samples previously measured by RBS analyses to have a mean surface mass around 260 µ g cm 2 . With polychromatic pink beam a close look at the undulator spectrum reveals a contribution of photons of higher energy harmonics at 16.9, 19.7, and 22.4 keV, which are greatly reduced by the lens and 10- µ m pinhole assembly installed before the sample. The
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