Chemical heterogeneity in cell death: Combined synchrotron IR and fluorescence microscopy studies of single apoptotic and necrotic cells

Abstract

The combination of synchrotron IR microspectroscopy and fluorescence microscopy has led to the identification of specific IR signatures of apoptosis and necrosis at a single cell level. Apoptosis was induced by treatment of Fas+ tumor cell lines with anti-Fas monoclonal antibodies. Detection of the early and late stages of apoptosis was performed using conjugated annexin V-fluorescein isothiocyanate (AV-FITC) and propidium iodide. Very early cellular changes were detected by IR before externalization of phosphatidylserine and AV-FITC labeling, and they were probably linked to DNA unwinding. The IR signals at 1044, 1177, and 1222 cm(-1), as well as an intensity variation in the CHx stretching region, are the main signature changes of early and late apoptosis, in line with the hypothesis of DNA fragmentation. The increased intensity of the CHx stretching bands of the lipids was observed only at an early stage of apoptosis. Changes in the relative intensity of CH3 and CH2 stretching accompany this increased intensity, suggesting changes in the relative amount and/or type of lipids concomitant with an increased lipid content. Finally, necrotic cells were characterized by marked changes in their chemical composition because several new vibrational features were observed.

Infrared spectral histopathology (SHP): a novel diagnostic tool for the accurate classification of lung cancer

“…The effect of necrosis on the observed vibrational spectra was first reported by Jamin, et al; 48 in these spectra, a strong shoulder of the amide I peak at ca 1630 cm À1 was reported (in fact, this shoulder was reported to have higher intensity than the 'main' amide I peak at 1655 cm À1 ). The large spectral changes observed for necrosis indicate major changes in the protein composition of necrotic cells, as the 1630 cm À1 peak is associated with unfolded and precipitated proteins.…”

“…The necrotic tissue spectra could be distinguished from non-necrotic cancer locations because necrosis induces a significant spectral change that has been detected and described very early in research efforts to detect disease by IR spectral methods; see below. 48 Classification Results/Pixel Spectrum Based The first classification scheme used the 106 000 annotated spectra, which were separated into completely independent training and test sets; see Table 1. According to the discussion in the previous paragraph, the top level ANN (ANN level 1, see Table 2) was trained to distinguish NORMAL from NOT NORMAL spectra (equivalent to CANCER vs NOT CAN-CER).…”

Chemical composition of the graphitic black carbon fraction in riverine and marine sediments at sub-micron scales using carbon X-ray spectromicroscopy

“…A detailed characterization of the GBC fraction of the BC pool in a variety of sedimentary environments may help further resolve the relative contributions of the modern and ancient sources to these environments. (Mitchell 2001;Sommer and Franke 2002;Jamin et al 2003). X-ray microscopic techniques are particularly promising in the investigation of recalcitrant particles, since they have small resolution limits (Jacobsen and Kirz 1998).…”

In vivo photothermal flow cytometry: Imaging and detection of individual cells in blood and lymph flow

“…In particular, to understand the mechanism of the development of metastasis, it is important to monitor tumor cell migration and interaction with other cells [Condeelis and Segall, 2003;Yamamoto et al, 2004;Tozer et al, 2005]. And finally, in vivo study of apoptosis with advanced molecular imaging is crucial for understanding human metabolic and immune system function [Brauer, 2003;Jamin et al, 2003].…”