Exp Neurobiol.  2008 Jun;17(1):17-24. 10.5607/en.2008.17.1.17.

Optimization of Wet Fixation Methods for AFM Imaging of Human Fibroblast Cells

• Lee GJ
• Uhm YK
• Eo YH
• Park JH
• Lim JE
• Jo TH
• Kim BS
• Choi SK
• Oh BS
• Lee MH
• Park HK

• KMID: 2165278
• DOI: http://doi.org/10.5607/en.2008.17.1.17

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.

Keyword

AFM; wet fixation method; fixative; human fibroblast cell

MeSH Terms

Artifacts
Cytoplasm
Fibroblasts
Formaldehyde
Glutaral
Humans
Microscopy, Atomic Force
Water
Formaldehyde
Glutaral
Water