The power of a microscope is measured by its resolution—the ability to clearly distinguish between two points that are very close together. To determine which microscope holds the title for the highest resolution, it is necessary to move beyond simple magnification and explore the physical limits imposed by the nature of light and matter. The highest resolution is achieved by abandoning traditional optical principles in favor of methods that probe the sample at the atomic scale.
The Physics of Resolution Limits
The resolution of any microscope using visible light is fundamentally constrained by the wave nature of light, a limitation known as the diffraction limit. This principle dictates that it is impossible for a lens to resolve objects separated by a distance smaller than half the wavelength of the light used for imaging. Since visible light has wavelengths ranging from approximately 400 to 700 nanometers, the theoretical limit for a conventional light microscope is roughly 200 nanometers.
Even with the highest quality lenses, the wave properties of light prevent the visualization of structures smaller than this 200-nanometer threshold. Structures significantly smaller than 200 nanometers, such as organelles or large molecules, appear as blurred discs under standard light microscopy. To see objects at the molecular or atomic level, a different form of illumination must be employed.
High Resolution Through Electron Beams
Achieving higher resolution involves switching from photons to electrons as the imaging source. This is effective because the de Broglie wavelength of a high-speed electron is thousands of times shorter than the wavelength of visible light. A shorter wavelength translates directly to a smaller diffraction limit and a much higher potential resolution. Electron microscopes utilize magnetic lenses instead of glass lenses to focus the electron beam onto the sample.
There are two primary types of electron microscopes. The Scanning Electron Microscope (SEM) scans a focused electron beam across a sample’s surface and collects the scattered electrons to create a detailed image of the topography. The SEM excels at visualizing surface features with a resolution typically in the low nanometer range.
The Transmission Electron Microscope (TEM) achieves significantly higher resolution by passing the electron beam directly through an ultrathin sample. Modern TEM instruments can achieve a resolution approaching 0.5 Ångströms (0.05 nanometers), precise enough to resolve individual columns of atoms. Electron microscopy requires the sample to be placed in an extremely high vacuum, and biological samples often require chemical fixation, which limits their use for studying living processes.
Probing the Atomic Surface
The absolute highest resolution is attained not by using beams, but by physically or electronically probing the surface of a material at the atomic scale. These instruments, known as Scanning Probe Microscopes (SPMs), include the Scanning Tunneling Microscope (STM) and the Atomic Force Microscope (AFM). These techniques bypass the diffraction limits entirely because they do not rely on a beam’s wavelength.
The Scanning Tunneling Microscope (STM) operates based on the quantum mechanical phenomenon of tunneling. When an extremely sharp, conductive tip is brought close to a conductive sample surface, a small voltage creates a tunneling current between the tip and the sample. This current is highly sensitive to the distance, allowing the instrument to map the surface atoms. Specialized STM instruments can achieve a lateral resolution of up to 0.1 nanometers and a vertical resolution of 0.01 nanometers, enabling the visualization of individual atoms.
The Atomic Force Microscope (AFM) overcomes the STM’s limitation of requiring a conductive sample. The AFM uses an ultrasharp tip mounted on a flexible cantilever to sense the physical forces between the tip and the sample surface. As the tip scans, these forces cause the cantilever to deflect, which is measured by a laser. This method allows the AFM to map the topography of insulators, polymers, and biological materials with atomic-scale precision, often achieving sub-nanometer resolution.
Breaking the Light Barrier
While the highest absolute resolution belongs to probe-based microscopes, a separate class of techniques has revolutionized biological imaging by breaking the light limit. These methods are collectively known as Super-Resolution Microscopy (SRM) or nanoscopy. They are distinct because they still use light, making them suitable for viewing dynamic processes in living cells.
Techniques such as STimulated Emission Depletion (STED) and Stochastic Optical Reconstruction Microscopy (STORM) use optical or computational tricks to localize fluorescent molecules with greater precision. STED uses a second, donut-shaped laser beam to deplete the fluorescence signal around the center of the excitation spot, shrinking the area from which light is emitted. STORM and Photoactivated Localization Microscopy (PALM) activate only a sparse subset of fluorescent molecules, allowing the precise location of each molecule to be determined computationally.
These super-resolution methods push the resolution of optical imaging down to the range of 5 to 20 nanometers, a significant improvement over the 200-nanometer diffraction limit. This level of detail is sufficient to resolve the fine structure of cellular components. Although this resolution does not compete with the sub-Ångström capabilities of electron or probe microscopes, SRM provides high spatial resolution within a native, wet, and living environment.