Table 1.
Technique | AFM | Super-resolution fluorescence microscopy (TIRF, PALM, STORM) | Electron microscopy (TEM) | |
Topographic imaging | SMFS | |||
Resolution | ≈50 nma | ≈20 nm | ≈20–50 nm | ≈1-10 nm |
Live cell | Yes | Yes | Yes | No |
Time for sample preparation | 10 minutes | 1 dayb | 1-3 hours | 2-5 days |
Time for image acquisition | ≈5 minutes | 25-50 minutes | ≈5 minutes | 5-10 minutes |
Image processing | 5 minutes | A few hours | Up to 24 hours | 30 minutes |
Simultaneous detection of different proteins | No | No | Yes | Yesc |
Protein tracking | No | No | Yes | No |
Cost of equipment | €150,000-250,000 | €250,000-500,000 | €≈500,000 | |
Operational costs | Low | Moderate | Moderate | High |
Advantages | Localization and force response of single proteins; protein unfolding; dynamic processes; various environments (temperature, buffer, etc.) | Spatial-temporal resolution; only basic genetic manipulations required; high sample throughput; study of live protein interactions and their quantification | Imaging of the cell ultrastructures at very high resolution | |
Disadvantages | Only the cell surface is analyzed; only a single cell at a time; slow temporal resolution; various sources of artifacts like cell or tip alteration | Frequently a bad signal-to-noise ratio; photobleaching; no information on physical properties of proteins; tendency of fluorescent protein tags to multimerize (i.e. interaction artifacts) | Fixation artifacts; no access to dynamics; no information on physical properties of proteins |
Only whole cell analyses are considered, i.e. purified proteins or membranes are not included. TIRF, total internal reflection fluorescence; PALM, photoactivated localization microscopy; STORM, stochastic optical reconstruction microscopy; TEM, transmission electron microscopy.
In mammalian cells (a 10-nm resolution can be routinely achieved on microbial cells).
Including tip preparation.
Indirect; e.g. gold beads of different sizes.