Table 1.

Comparison of high-resolution techniques for imaging single proteins in cells

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.

^aIn mammalian cells (a 10-nm resolution can be routinely achieved on microbial cells).

^bIncluding tip preparation.

^cIndirect; e.g. gold beads of different sizes.