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. Subject category: Mass spectrometry.
Institute of Nuclear Physics, Polish Academy of Sciences, Radzikowskiego 152, 31-342 Cracow, Poland.
Justyna
J
Bobrowska
Institute of Nuclear Physics, Polish Academy of Sciences, Radzikowskiego 152, 31-342 Cracow, Poland.
Joanna
J
Pabijan
Institute of Nuclear Physics, Polish Academy of Sciences, Radzikowskiego 152, 31-342 Cracow, Poland.
Joanna
J
Wiltowska-Zuber
Marian Smoluchowski Institute of Physics, Jagiellonian University, Lojasiewicza 11, 30-348 Cracow, Poland.
Benedykt R
BR
Jany
Marian Smoluchowski Institute of Physics, Jagiellonian University, Lojasiewicza 11, 30-348 Cracow, Poland.
Franciszek
F
Krok
Marian Smoluchowski Institute of Physics, Jagiellonian University, Lojasiewicza 11, 30-348 Cracow, Poland.
Kamil
K
Awsiuk
Marian Smoluchowski Institute of Physics, Jagiellonian University, Lojasiewicza 11, 30-348 Cracow, Poland.
Jakub
J
Rysz
Marian Smoluchowski Institute of Physics, Jagiellonian University, Lojasiewicza 11, 30-348 Cracow, Poland.
Andrzej
A
Budkowski
Institute of Nuclear Physics, Polish Academy of Sciences, Radzikowskiego 152, 31-342 Cracow, Poland. Electronic address: Malgorzata.Lekka@ifj.edu.pl.
Malgorzata
M
Lekka
D045744:Cell Line, Tumor; D006801:Humans; D013048:Specimen Handling / Q000379:methods*; D018629:Spectrometry, Mass, Secondary Ion / Q000379:methods*
AFM; ESEM; Melanoma cells; Sample preparation protocol; ToF SIMS