Cameras have a fixed observation field and cannot scan the full FOV of the microscope optics. Consequently, the field must be reduced (by insertion of additional optical components) so that the image can be projected onto the camera sensor without losses.
In scanning microscopes, like true confocal laser scanners, the FN indicates the maximum area over which the sample can be scanned, but the actual scan process must be set with the correct parameters or resolution may be lost. This condition usually means smaller fields should be observed or very large image formats used. To be able to compare these restrictions, a modified field number (RFN) is introduced. RFN refers to the maximum resolved field.
Field Number (FN)
The field number (FN) in microscopy is defined as the diameter of the area in the intermediate image plane that can be observed through the eyepiece. A field number of, e.g., 20 mm indicates that the observed sample area after magnification by the objective lens is restricted to a diameter of 20 mm. If the objective lens has a magnification of, say, 40x with an FN of 20 mm, a sample area with a diameter of 500 µm can be observed.
With dfield is the diameter of the observed sample area and Mag is the magnification factor of the objective lens.
Usually, the magnification value is engraved on the lens barrel and the field number is engraved on the eyepiece barrel. With that information, you can always calculate the diameter of the FOV you are currently observing:
For widefield microscopes, the FN is set by the diameter of a metal diaphragm in the eyepiece. Its size is set to ensure good image quality within the observed field, as all optical systems cause increasing optical aberration far from the center of the lens. Nevertheless, the definition of “good quality” is only standardized for the flatness of field (1), leaving room for discussion about other aberrations.
In confocal scanning microscopes, an intermediate image as such is actually not generated. In fact, the pinhole is located in the intermediate image plane. The intensity as a function of time that passes the pinhole, codes for the image information. These intensity changes are subsequently used to reconstruct the image by distribution of the appropriate time-intensity segments into a frame store. The frame store is filled synchronously to the position of the focus spot in the sample. The frame has a predefined size of x times y picture elements (pixels). The tuple (x,y) is called the scan-format or sometimes scan-resolution (not to be confused with the optical resolution (2)).
The calculated FN for a confocal microscope is, therefore, the scanned field size of the sample multiplied by the magnification. The intermediate image does not appear during imaging. By tuning the scanning amplitude, the observed field can be changed from a maximum down to zero (3). Zero amplitude means that a single, fixed spot in the sample is observed, as is required for fluorescence correlation spectroscopy (