Articles

Article

Original

Exp Neurobiol 2008; 17(1): 17-24

Published online June 30, 2008

© The Korean Society for Brain and Neural Sciences

Optimization of wet fixation methods for AFM imaging of human fibroblast cells

Gi Ja Lee1,2, Yoon Kyung Uhm3,Yun-Hye Eo1,2, Ji Hye Park1,2, Ji Eun Lim1,2, Tae Ho Jo4, Bum Shik Kim2, Seok Keun Choi2, Berm Seok Oh1,2,5, Mu Hyoung Lee4 and Hun Kuk Park1,2,5,*

1Dept. of Biomedical Engineering, School of Medicine, 2Healthcare Industry Research Institute, 3Dept. of Pharmacology, School of Medicine, 4Dept. of Dermatology, School of Medicine, 5Program of Medical Engineering, Kyung Hee University, Seoul 130-702, South Korea

Correspondence to: *To whom correspondence should be addressed.
TEL: 82-2-961-0290, FAX: 82-2-961-5515
e-mail: sigmoidus@khu.ac.kr

Abstract

We investigated the effect by the chemical fixative on human fibroblast cells (HFCs) in order to make nano-scale images using by the atomic force microscopy (AFM). The cell fixation needed to be optimized as prerequisite step for the preparation before analysis. AFM imaging after optimal wet fixation can provide practical, simple and fast technique for scanning living cells. In this study, AFM images - topography and amplitude - and the optic images of HFCs which were fixed with phosphate buffered saline (PBS), 2:1 ethanol:acetic acid, 4% glutaraldehyde and 37% formaldehyde were compared respectively. The final effect by washing with PBS or distilled water (D.W.) was examined after 4% glutaraldehyde fixation. To determine the optimal fixation method for HFCs, we performed quantitative and qualitative analysis by the height profile, the presence of artifacts and the morphology of well-conserved fibroblastic topography image by AFM. From AFM image which showed fibroblastic cellular morphology and differential height value of cytoplasm (670±47 nm, n=10) and nucleus (847±32 nm, n=10) in HFCs, we proposed that wet fixation by 4% glutaraldehyde, followed by final washing with PBS, could be the most suitable preparation for AFM imaging of HFCs, which enable us to approach easily on living cells with the least shrinkage.

Keywords: AFM, wet fixation method, fixative, human fibroblast cell