The best resolution achievable by a light microscope is approximately 200 nm (nanometers) laterally and 500 nm axially. This limit is due to the diffraction of light, a fundamental property that affects how we can see small objects using lenses.
Understanding Resolution in Light Microscopy
Resolution refers to the ability of a microscope to distinguish between two closely spaced objects as distinct entities. A higher resolution means you can see finer details. Light microscopes are bound by the laws of physics, specifically the diffraction of light, which limits their resolving power.
- Lateral Resolution: This refers to the resolution in the x-y plane (side to side). The theoretical limit is about 200 nm.
- Axial Resolution: This refers to the resolution in the z-axis (depth). It's typically lower than lateral resolution, around 500 nm.
Factors Affecting Resolution
Several factors influence the actual resolution achieved in practice:
- Wavelength of Light: Shorter wavelengths of light provide better resolution. This is why techniques like UV microscopy can achieve slightly better resolution than standard brightfield microscopy.
- Numerical Aperture (NA) of the Objective Lens: A higher NA lens collects more light, leading to improved resolution. Immersion oil objectives have higher NAs than dry objectives.
- Sample Preparation: Proper sample preparation is crucial. Artifacts or poor staining can obscure details and reduce the effective resolution.
- Microscope Alignment: Correct alignment of the microscope's optical components is essential for optimal performance.
Beyond the Diffraction Limit
While conventional light microscopy is limited by diffraction, advanced techniques have been developed to overcome this barrier, often referred to as "super-resolution" microscopy. Some examples include:
- Structured Illumination Microscopy (SIM): Uses patterned light to improve resolution.
- Stimulated Emission Depletion (STED) Microscopy: Uses lasers to selectively deactivate fluorescence, allowing for finer details to be resolved.
- Photoactivated Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM): These techniques use fluorescent molecules that can be switched on and off, allowing for precise localization and reconstruction of high-resolution images.
These super-resolution techniques can achieve resolutions significantly better than the diffraction limit, sometimes down to 20-30 nm. However, they often require specialized equipment, sample preparation, and analysis methods.
