Resolving Power of Microscope:

For microscopes, the resolving power is the inverse of the distance between two objects that can be just resolved. This is given by the famous Abbe’s criterion given by Ernst Abbe in 1873 as

△d=λ/2nsinθ

Resolving power =1/△d=2nsinθ/λ

Wherenis the refractive index of the medium separating object and aperture. Note that to achieve high resolutionn sin θmust be large. This is known as the Numerical aperture.

Thus, for good resolution:

sin θmust be large.To achieve this, the objective lens is kept as close to the specimen as possible.

A higher refractive index (n) medium must be used.Oil immersion microscopes use oil to increase the refractive index. Typically for use in biology studies this is limited to 1.6 to match the refractive index of glass slides used. (This limits reflection from slides). Thus the numerical aperture is limited to just 1.4-1.6. Thus, optical microscopes (if you do the math) can only image to about 0.1 micron. This means that usually organelles, viruses and proteins cannot be imaged.

Decreasing the wavelength by using X-rays and gamma rays.While these techniques are used to study inorganic crystals, biological samples are usually damaged byx-raysand hence are not used.

The limit set by Abbe’s criterion for optical microscopy cannot be avoided. However, using different fluorescence microscopy techniques the Abbe’s limit can be circumvented. Stefan Hell used a technique called Simulated Emission Depletion (STED) and the duo Eric Betzig and W.E. Moerner used super imposed images using green fluorescent proteins to bypass the resolution limit and obtain optical images in never before seen resolution. All three were awarded the 2014 Nobel Prize in Chemistry for their pioneering work.