Question

'' Which parameters determine the optical resolution in an ideal optical microscope? Given this dependence why it is problematic to use UV light in optical microscopy, in particular in live specimen?''

Please answer well so I can understand, Thank you so much.  

Answer 1

Optical resolution describes the ability of an imaging system to resolve detail in the object that is being imaged. In other words, this is the minimum distance at which two distinct points of a specimen can still be seen - either by the observer or the microscope camera - as separate entities.Ok

Now the parameters, determining are:-

The numerical aperture (NA) is related to the refractive index (n) of a medium through which light passes as well as the angular aperture (α) of a given objective (NA= n x sin α).

But there are more factors too,

n a microscope system in which all of the optical components are correctly aligned, have a relatively high NA value and are working harmoniously with each other. Resolution is also related to the wavelength of light which is used to image a specimen; the light of shorter wavelengths are capable of resolving greater detail than longer wavelengths.

‘Abbe’s Diffraction Limit’, ‘Airy Discs’ and the ‘Rayleigh Criterion. These three mathematical concepts need to be taken in consideration while dealing with resolutions.

An Airy Disc is the optimally focussed point of light which can be determined by a circular aperture in a perfectly aligned system limited by diffraction.

The diffraction pattern is determined by the wavelength of light and the size of the aperture through which the light passes. The central point of the Airy Disc contains approximately 84% of the luminous intensity with the remaining 16% in the diffraction pattern around this point. There are of course many points of light in a specimen as viewed with a microscope, and it is more appropriate to think in terms of numerous Airy Patterns as opposed to a single point of light as described by the term ‘Airy Disc’.

In order to increase the resolution (d=λ/2 NA), the specimen must be viewed using either shorter wavelength (λ) light or through an imaging medium with a relatively high refractive index or with optical components which have a high NA (or, indeed, a combination of all of these factors).

even taking all of these factors into consideration, the limits in a real microscope system are still somewhat limited due to the complexity of the whole system, transmission characteristics of glass at wavelengths below 400 nm and the achievement of a high NA in the complete microscope. Lateral resolution in an ideal light microscope is limited to around 200 nm, whereas axial resolution is around 500 nm

Shorter the wavelength higher the transmission

The short wavelength of UV light helps to improve the image resolution beyond the diffraction limit of optical microscopes using normal white light.

The response of the sample to UV light is greater than that achieved by use of white light, in respect to the surroundings. As a result, there is an increased contrast in the image created by the microscope so that it is easier to view the samples.

The short wavelength of UV light helps to improve the image resolution beyond the diffraction limit of optical microscopes using normal white light.

The response of the sample to UV light is greater than that achieved by use of white light, in respect to the surroundings. As a result, there is an increased contrast in the image created by the microscope so that it is easier to view the samples.

The glass lenses usually used in optical microscopes created a cloudy or unclear image when used at the shorter wavelength of UV light.Also the living speciemen may face issues due to UV rays