A focusing device comprising: an imaging device provided with a predetermined image forming surface separate from a main image forming surface of an object lens on which phase contrast images of an image formed on the main image forming surface are formed; a processor (30, 46); and a memory device operatively coupled to the processor (30, 46), the memory device storing instructions that cause the processor, in cooperation with the memory device, to: (a) generate a subject image associated with a sample on the main image forming surface of a main imaging device and a plurality of phase contrast images associated with the sample on a predetermined imaging device being separate from the main imaging device; (b) determine distances between corresponding pixels of: (i) a first phase contrast image associated with the sample; and (ii) a second phase contrast image associated with the sample; and (c) determine a focusing position of an object lens based on the determined distances, wherein the instructions, when executed by the processor, cause the processor (30, 46) further to: (d) determine a value corresponding to a difference between a maximum value of distance and a minimum value of distance; (e) in response to the value being larger than a predetermined threshold, for the lens, divide an imaging area into a plurality of layers; and (f) for each of the layers, determine a separate focusing position of the object lens. an imaging device provided with a predetermined image forming surface separate from a main image forming surface of an object lens on which phase contrast images of an image formed on the main image forming surface are formed; a processor (30, 46); and a memory device operatively coupled to the processor (30, 46), the memory device storing instructions that cause the processor, in cooperation with the memory device, to: (a) generate a subject image associated with a sample on the main image forming surface of a main imaging device and a plurality of phase contrast images associated with the sample on a predetermined imaging device being separate from the main imaging device; (b) determine distances between corresponding pixels of: (i) a first phase contrast image associated with the sample; and (ii) a second phase contrast image associated with the sample; and (c) determine a focusing position of an object lens based on the determined distances, wherein the instructions, when executed by the processor, cause the processor (30, 46) further to: (d) determine a value corresponding to a difference between a maximum value of distance and a minimum value of distance; (e) in response to the value being larger than a predetermined threshold, for the lens, divide an imaging area into a plurality of layers; and (f) for each of the layers, determine a separate focusing position of the object lens. (a) generate a subject image associated with a sample on the main image forming surface of a main imaging device and a plurality of phase contrast images associated with the sample on a predetermined imaging device being separate from the main imaging device; (b) determine distances between corresponding pixels of: (i) a first phase contrast image associated with the sample; and (ii) a second phase contrast image associated with the sample; and (i) a first phase contrast image associated with the sample; and (ii) a second phase contrast image associated with the sample; and (c) determine a focusing position of an object lens based on the determined distances, wherein the instructions, when executed by the processor, cause the processor (30, 46) further to: (d) determine a value corresponding to a difference between a maximum value of distance and a minimum value of distance; (e) in response to the value being larger than a predetermined threshold, for the lens, divide an imaging area into a plurality of layers; and (f) for each of the layers, determine a separate focusing position of the object lens.