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

• Published in: Analytical biochemistry Vol. 511; pp. 52 - 60
• Main Authors: Bobrowska, Justyna, Pabijan, Joanna, Wiltowska-Zuber, Joanna, Jany, Benedykt R., Krok, Franciszek, Awsiuk, Kamil, Rysz, Jakub, Budkowski, Andrzej, Lekka, Malgorzata
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.10.2016
• Subjects: AFM; Cell Line, Tumor; drying; ESEM; fluorescence; freezing; Humans; mass spectrometry; melanoma; Melanoma cells; microscopes; nitrogen; principal component analysis; Sample preparation protocol; Specimen Handling - methods; Spectrometry, Mass, Secondary Ion - methods; ToF SIMS; AFM; ToF SIMS; Sample preparation protocol; Melanoma cells; ESEM
• ISSN: 0003-2697; 1096-0309; 1096-0309
• DOI: 10.1016/j.ab.2016.06.011

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. Subject category: Mass spectrometry. •The protocol for preparation of samples with single cells for ToF SIMS experiments was proposed.•Preserved melanoma cell structure and morphology was visualized using AFM and ESEM.•ToF SIMS mass spectra analyzed by PCA differentiated between melanoma cell types.