The diffraction effects in a microscopic specimen become important when the separation between two points is

When the wavelength of light used is comparable with the separation between two points, the image of the object will be a diffraction pattern whose size will be 

$\theta = \frac{1.22\lambda }{D}$

$$\begin{gathered} \mathrm{where} \mathrm{\lambda } = \mathrm{wavelength} \mathrm{of} \mathrm{light} \mathrm{used} \\ \mathrm{D} = \mathrm{diameter} \mathrm{of} \mathrm{the} \mathrm{objective} \end{gathered}$$

Two objects whose images are closer than this distance, will not be resolved.