Microscope Resolution

 

Microscope resolution depends on the wavelength of light (λ) and the numerical aperture of the objective lens (NA). Use 550 nm for white light.

 

Lateral resolution = 0.61 λ / NA Objective

 

or if you include the condenser:    Lateral resolution = 1.22 λ / (NA Objective + NA Condenser)

 

Depth (Z) resolution  = 2 λ / NA squared

 

NA (numerical aperture) is a measure of the ability of the lens to gather light from a wide solid angle.

 

NA = n. Sin(α)

 

n = refractive index of immersion medium (air = 1, oil = 1.5, glycerine = 1.5, water = 1.3)

α = half the solid angle of light gathered by the lens (max  90 deg)

 

Cameras and Pixels

 

Image pixel size depends on the camera, C mount and objective lens: 

 

Image pixel size = CPS / (Mag * CM)

Camera field of view = (CPS * NPix) / (Mag * CM)

 

CPS = camera pixel size (typically 2 μm – 7 μm)

NPix = camera number of pixels in X and Y dimensions

Mag = objective lens magnification

CM = C mount magnification (typically 0.4x – 1x)

 

EyePiece

 

Field of view = Eyepiece Field Number (mm) / Objective Magnification

 

Camera Sampling

 

This graph shows you if the camera is over sampling or under sampling the microscope image. For example, if you are using an objective with a NA of 0.8 then the pixel size must be 0.2 microns or smaller. This can typically be achieved for a 20x objective with a 1.4 MPixel camera, i.e. it is surprising how few camera pixels are required to correctly sample the microscope image.