What analysis spot size is used for a Raman microscope?

The laser spot size is primarily defined by the laser wavelength and microscope objective being used.  The minimum achievable spot size is diffraction limited, according to the laws of physics and optics. 

Laser spot diameter = 1.22 λ /  NA

where λ is the wavelength of the laser, and NA is the numerical aperture of the microscope objective being used.  For example, with a 532 nm laser, and a 0.90/100x objective, the theoretical spot diameter will be 721 nm.

From this equation it can be seen that lower wavelength lasers offer high spatial resolution (e.g., a blue laser at 488nm will have a smaller spot size than an infra-red laser at 785nm if the same objective is used), as do high NA objectives (e.g., a 0.90/100x objective will give a smaller spot than a 0.55/50x objective).

A slightly modified equation yields the theoretical diffraction limited spatial resolution which is achievable using an optical microscope:

Spatial resolution = 0.61 λ  /  NA

For a 532 nm laser with a 0.90/100x objective this would predict a spatial resolution of 361 nm.  However, whilst this equation is applicable for standard light microscopy, the optical processes occurring during Raman microscopy are much more complex.  For example, scattering of the laser/Raman photons and interaction with interfaces in the sample can reduce this resolution.  Thus, typical Raman spatial resolution is often quoted as being in the order of 1 µm, whilst with ‘good’ samples spatial resolution approaching the diffraction limit can be achieved.

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