Investigating Living Cell Structure & Mechanics Using AFM & FM

Investigating Living Cell Structure and Mechanics Using Atomic Force and Fluorescence Microscopy:

Introduction:

In collaboration with the School of Biomolecular Sciences at Liverpool JMU and Université Claude Bernard, Lyon, the General Engineering Research Institute are involved in a project which aims to investigate the structural and mechanical properties of living human cells. The principle tools currently used in this research are the Asylum Research, Molecular Force Probe atomic force microscope couple to an inverted Olympus IX 50 (MFP-3D-IO) situated here in the General Engineering Institute and the semi-automated Olympus BX51 fluorescent microscope situated in the School of Biomolecular Sciences.

Basic Cell Structure:

The mammalian cell consists of a double layer lipid membrane surrounding a viscous fluid (cytosol), which contains a central nucleus. The structure and mechanical integrity of the cell is largely governed by a mesh-like structure known as the cytoskeleton which is dispersed throughout the cytosol and consists of three types of polymeric protein filaments:

• Actin Filaments
• Microtubules
• Intermediate Filaments

Figs. 1 and 2 below show the actin and microtubule network within human lung fibroblast cells imaged using fluorescence microscopy. The filaments have been labelled using fluorescent antibody-conjugates specific for each filament type.

 Figure 1: Actin network within a human lung fibroblast cell

 

Figure 2: Microtubule network within a human lung fibroblast cell

Cell Structure and Mechanical Properties:

Over the past twenty years the importance of cell structure and mechanics has become more prominent and has led to investigations to evaluate cell mechanics in terms of cell stiffness, elasticity, shear elasticity and viscoelasticity. This was mainly done using cell indentation techniques (i.e. cell poker) and shear measurements using magnetic tweezers. However, the past ten years has seen a rise in cell mechanical studies, which is largely due to the advent of the atomic force microscope.
Even so, much of the available data on cell mechanics is only qualitative, while much of the available quantitative data is incomplete and/or lacks analysis from an engineering perspective.

Atomic Force Microscope:

The atomic force microscope (AFM) is a valuable tool for biologists because of its ability to image and measure forces of biological samples in a physiologically stable environment. Initial work in our labs has focused on imaging living cells with the AFM which can be quite challenging, however when successful, images with nanometer resolution can be obtained which can provide accurate measurements on cell geometry, surface structure and morphological features and differences between cells. In contrast, by use of force versus distance curves forces in the Pico Newton (10-12) range can be measured (see fig 9)    

Figs. 3 and 4 below show an AFM deflection image and 3-D reconstructed height image of a living human lung epithelial cell (NCI H727 cancer cell line).

Figure 3: AFM deflection image of a living human lung epithelial cell  

        

Figure 4: 3-D reconstructed height image of a living human lung epithelial cell

Figs. 5 and 6 below show an AFM deflection image and 3-D reconstructed height image of a living human primary lung fibroblast cell (LL24 normal cell line).

Figure 5: AFM deflection image of a living human primary lung fibroblast cell

 

Figure 6: 3-D reconstructed height image of a living human primary lung fibroblast cell
   

Figs. 7 and 8 below show an AFM deflection image and 3-D reconstructed height image of a living human primary lung fibroblast cell (LL24 normal cell line) the same cell type as shown above in Figs. 5 and 6. The images show how even images of the same cell type can differ.

Figure 7: AFM deflection image of another living human primary lung fibroblast cell

 

Figure 8: 3-D reconstructed height image of another living human primary lung fibroblast cell

 

Figure 9: Diagram of AFM force curve taken on a hard substrate such as glass (1) and a cell (2). The difference between the two is a measure of cell indentation.

Future Project Aims:

This current project will aim to use biological, engineering and computing techniques and      
analysis in order to study the mechanical properties of live human cells. Using AFM the project will try to determine how cells behave mechanically when subjected to applied external forces. Currently, two theories exist to describe the cell behaviour in terms of a cell model. One theory suggests the cell displays largely viscoelastic properties while the other suggests the cell acts as a tensegrity structure having both tensional and compression structures (actin and microtubules). In particular, our research will focus on the role of the cell cytoskeleton on mechanical integrity of both normal cells and cells of a pathological nature. Different cell types will also be investigated to look for differences in mechanical properties between cells. The data obtained throughout this study will provide new and valuable information about cell mechanics in general while contributing to the existing data, particularly that on cell model theory.

Mark Murphy.