Mid-Infrared Spectroscopy and Microscopy of Subcellular Structures in Eukaryotic Cells with Atomic Force Microscopy - Infrared Spectroscopy

Atomic Force Microscopy - Infrared (AFM-IR) spectroscopy allows spectroscopic studies in the mid-infrared spectral region with a spatial resolution better than 50 nm. We show that the high spatial resolution can be used to perform spectroscopic and imaging studies at the subcellular level in fixed eukaryotic cells. We collect AFM-IR images of subcellular structures that include lipid droplets, vesicles and cytoskeletal filaments, by relying on the intrinsic contrast from IR light absorption. We also obtain AFM-IR absorption spectra of individual structures. Most spectra show features that are recognizable in the IR absorption spectra of cells and tissue obtained with FTIR technology, including absorption bands characteristic of phospholipids and polypeptides. We also observe sharp spectral features that we attribute to the nonlinear photothermal response of the system and we propose that they can be used to perform sensitive spectroscopy on the nanoscale

Protocol of single cells preparation for time of flight secondary ion mass spectrometry

There are several techniques like time of flight secondary ion mass spectrometry (ToF SIMS) that require a special protocol for preparation of biological samples, in particular, those containing single cells due to high vacuum conditions that must be kept during the experiment. Frequently, preparation methodology involves liquid nitrogen freezing what is not always convenient. In our studies, we propose and validate a protocol for preparation of single cells. It consists of four steps: (i) paraformaldehyde fixation, (ii) salt removal, (iii) dehydrating, and (iv) sample drying under ambient conditions. The protocol was applied to samples with single melanoma cells i.e. WM115 and WM266-4 characterized by similar morphology. The surface and internal structures of cells were monitored using atomic force, scanning electron and fluorescent microscopes, used to follow any potential protocol-induced alterations. To validate the proposed methodology for sample preparation, ToF SIMS experiments were carried out using C60+ cluster ion beam. The applied principal component analysis (PCA) revealed that chemical changes on cell surface of melanoma cells were large enough to differentiate between primary and secondary tumor sites