Maximum resolving power and depth of focus for a visual microscope objective focal length (mm) numerical aperture (N.A).
The choice of the objective focal length is imposed by the required field-of-view (FOV) in the direction parallel to the slit.
As a result, excitation spots can be located within all the available excitation field available, i.e. <img src="http://journals.plos.org/plosone/article/asset?id=info?doi/10.1371/journal.pone.0009431.e001.PNG" class= inline-graphic"/>, where d is the spatial frequency corresponding to the maximum deflection angle from LCOS-SLM and fobj the objective focal length.
Solving the coupled lens equations for this system we obtain, <img src="http://journals.plos.org/plosone/article/asset?id=info?doi/10.1371/journal.pone.0016772.e022.PNG" class= inline-graphic"/> where fO is the objective effective focal length, fT is the tube lens focal length, and L is tube length.
The overall magnification is given by the objective, the focal length f1 of the tube lens L1, the position and the focal length f2 of lens L2.
In the sample arm, the beam is scanned on the sample in telecentric geometry using a galvanometer based optical scanner (Cambridge Technologies, model # 6220) and is focused by an objective lens (focal length = 60 mm, working distance = 56 mm, diameter = 25.4 mm) which has two achromatic doublets with focal lengths 100 mm and 150 mm (Thorlabs) separated by 5 mm.
Once you have the focal lengths for both of your lenses, solving is easy — just find the ratio by dividing the objective's focal length by the eyepiece's.
A pair of micro objectives (25× magnification, 0.4 numerical aperture) are used in the sample arm, thus the transmissive confocal system can be described as focusing light with one objective onto the focal plane, then reimaging by a second objective with same focal length [ 16].
For example, if the objective has a focal length of 254 cm (100 inches) and the eyepiece has a focal length of 2.54 cm (1 inch), then the magnification will be 100.
eNA was defined as the equivalent NA of an objective of same focal length illuminated with a constant intensity profile and providing the same in-plane resolution.
The confocal configuration of the probe adopted throughout the present experiments corresponded to a ×100 objective; numerical aperture, focal length, and pinhole diameter were fixed as 0.6, 7.6 mm, and 100 μm, respectively.
