Microwave atomic force microscope: MG63 osteoblast-like cells analysis on nanometer scale

Abstract

In this paper, we report a non-invasive and non-destructive probing method for analyzing the MG63 osteoblast-like cells. High frequency microwave atomic force microscope (M-AFM) can be used to measure the surface topography and microwave image of MG63 cells simultaneously in one scanning process. Under the frequency modulation AFM mode, the M-AFM probe tip can scan above the cell surface, maintaining a constant stand-off distance and the created lateral forces were small enough as not to sweep away or deform the fragile biomolecules. By analyzing the results, quantification such as, the number and distribution of organelles and proteins of MG63 cells as well as their dimension and electrical property information can be characterized. The unique potentials of that M-AFM imaging biological substrates with no damaging manner and nanometer scale resolution, while the original structure and function of the biomolecules during the investigation are preserved, make this technique very attractive to biologists.

Appendix

Cell culture: MG-63 Osteoblast-like cells (cell bank), which has a number of characteristic features of osteoblasts, was used in this study. MG63 cells were cultured in Dulbbeco’s modified Eagle’s medium (DMEM, Gibco). 10 % fetal bovine serum (FBS) and 1 % penicillin/streptomycin were added to culture media. MG63 cells were seeded in 25 cm² culture flasks (Becton–Dickinson Labware, USA) and kept in humidified incubator (SANYO, Japan) at 37 °C under 5 % CO₂ atmosphere. The culture medium was changed every 3 days. After reaching confluence, cells were harvested with 0.25 % trypsin/0.02 % EDTA (Takara Bio Inc., Japan).

Sample preparation for M-AFM and SEM: to prepare MG63 sample for M-AFM detection, the cells were cultured for 2 days and rinsed with PBS, and then fixed with 2.5 % glutaraldehyde (Wako, Japan) in PBS for 1 h at room temperature. After thorough washing with PBS, the cells were dehydrated in ethanol graded series (50, 60, 70, 80, 90, 95 and 100 %) for 15 min each and air-dried at room temperature. For SEM observation, the as prepared sample was sputter-coated with gold with 100 nm (Canon, E-200S, Japan) and cell morphology was examined using SEM (JEOL 7000FK, Japan).

Immunofluorescence staining: After MG63 cells were cultured on the cover glass for 2 days, the surfaces were rinsed with PBS and the cells attached on the surfaces were fixed with 4 % paraformaldehyde in PBS for 20 min at room temperature. After thorough washing with PBS for three times, the cells on the substrate were permeablized with 0.25 % Triton X-100 at room temperature for 30 min. Then, samples were rinsed with PBS for three times and stained with 0.5 μM Alexa Fluor 488 conjugated phalloidin at room temperature for 2 h. The nuclei of MG63 were stained with 10 µg/ml DAPI and counterstained with mounting medium at room temperature for 5 min, respectively. The stained samples were finally observed with confocal CLSM (Nikon, Japan).