Label-free Raman observation of cytochrome c dynamics during apoptosis

We performed label-free observation of molecular dynamics in apoptotic cells by Raman microscopy. Dynamic changes in cytochrome c distribution at the Raman band of 750 cm-1 were observed after adding an apoptosis inducer to the cells. The comparison of mitochondria fluorescence images and Raman images of cytochrome c confirmed that changes in cytochrome c distribution can be distinguished as release of cytochrome c from mitochondria. Our observation also revealed that the redox state of cytochrome c was maintained during the release from the mitochondria. Monitoring mitochondrial membrane potential with JC-1 dye confirmed that the observed cytochrome c release was associated with apoptosis

Vibrational spectroscopic analysis of peripheral blood plasma of patients with Alzheimer’s disease

Using Raman and infrared spectroscopy, we monitored spectral changes occurring in the blood plasma of patients with Alzheimer’s disease (AD) in relation to healthy controls. The protein secondary structure as reflected by amide I band involves β-sheet enrichment, which may be attributable to Aβ peptide formation and to increasing proportion of the globulins that are β-sheet rich. Likewise, the behavior of the infrared 1200–1000–cm−1 region and the Raman 980–910- and 450–400-cm−1 regions can be explained in terms of the said plasma composition change. Further, the 744-cm−1 Raman band from healthy control plasma shows frequency upshifting in the course of AD, which may be generated by the platelets collected in blood plasma. Linear discrimination analysis and receiver operating characteristic (ROC) analysis have been used to distinguish between patients with AD and age-matched healthy controls with a diagnostic accuracy of about 94 %.Ministerio de Ciencia e Innovación de EspañaMinisterio de Economía y Competitividad de EspañaSección Deptal. de Química Orgánica (Óptica y Optometría)Fac. de Óptica y OptometríaTRUEpu

Mid-infrared metabolic imaging with vibrational probes.

Understanding metabolism is indispensable in unraveling the mechanistic basis of many physiological and pathological processes. However, in situ metabolic imaging tools are still lacking. Here we introduce a framework for mid-infrared (MIR) metabolic imaging by coupling the emerging high-information-throughput MIR microscopy with specifically designed IR-active vibrational probes. We present three categories of small vibrational tags including azide bond, 13C-edited carbonyl bond and deuterium-labeled probes to interrogate various metabolic activities in cells, small organisms and mice. Two MIR imaging platforms are implemented including broadband Fourier transform infrared microscopy and discrete frequency infrared microscopy with a newly incorporated spectral region (2,000-2,300 cm-1). Our technique is uniquely suited to metabolic imaging with high information throughput. In particular, we performed single-cell metabolic profiling including heterogeneity characterization, and large-area metabolic imaging at tissue or organ level with rich spectral information