Your molecular biology lab studies proteins, and you're frustrated because your microscopes can't quite resolve crystallized proteins. A sales rep touts the advantages of an expensive microscope using 200 -nm ultraviolet light, saying you'll be able to resolve structures less than half the size that's resolvable with your optical microscopes. Is the sales rep correct?

When examining the minutiae of biological specimens, optical microscopes are an invaluable tool, but they have their limitations.

At the core of an optical microscope's limitations is its resolving power, the microscope's ability to distinguish between two points as separate entities. This power largely depends on the wavelength of light used for imaging. As visible light typically ranges from 400 to 700 nanometers (nm) in wavelength, structures that are smaller than the wavelength become challenging, if not impossible, to resolve distinctly.

What does this mean practically? If a researcher tries to view objects closer together than about half the wavelength of visible light, they would appear as a single blurred image rather than two distinct points. This limitation is a major hurdle when studying small structures like protein crystals. To go beyond this limit, researchers must turn to other types of microscopy, such as electron or ultraviolet microscopy.

Stepping beyond the boundaries imposed by visible light, an ultraviolet (UV) microscope makes use of shorter wavelengths that fall in the range of 10 to 400 nm.

By employing ultraviolet light, usually around the 200 nm mark, UV microscopes can resolve structures much smaller than those visible under an optical microscope. This is particularly advantageous for biomedical research as it allows for the observation of finer details within cells, including proteins and other complex molecules.

The sales representative's claims that a microscope utilizing 200 nm UV light can resolve structures less than half the size visible with an optical microscope hold some truth. However, the extent to which the resolution improves must be accurately quantified. It is essential to understand that while a UV microscope offers significantly enhanced resolution, it may require special sample preparation and safety measures due to the potential hazards of UV light exposure.

To quantify resolving power, scientists often refer to the Rayleigh criterion, a formula used to determine the minimum distance between two points that a microscope can discern as separate.

The criterion states that two points are resolved when the principal maximum of the diffraction pattern of one image coincides with the first minimum of the other. In simpler terms, one often uses the formula \( d = 1.22 \frac{\lambda}{NA} \) where \( d \) is the minimum resolvable distance, \( \lambda \) is the wavelength of light, and NA is the numerical aperture of the microscope lens.

When comparing the optical microscope with the ultraviolet microscope using the Rayleigh criterion, the utility of shorter wavelengths becomes clear. Shorter wavelengths, like the 200 nm UV light, allow for a smaller value of \( d \) and thus a greater resolving power. However, it's crucial to acknowledge that practical factors, such as lens quality and specimen preparation, can also affect the effective resolution achieved.