A method for calibrating an imaging system for imaging biological or chemical samples, the method comprising: for each of a plurality of initial effective distances from an optical component of the imaging system to a calibration target at a sample location, identifying an initial optimal focus setting of the optical component, wherein different initial effective distances correspond to different initial optimal focus settings of the optical component; storing first data for the imaging system to determine a first functional approximation that is derived from the initial optimal focus settings of the optical component for the initial effective distances, the first data being stored in at least one computer readable medium that is adapted to be communicably coupled with the imaging system, wherein the first functional approximation is operable for calculating a new optimal focus setting of the optical component for a new effective distance that is not one of the initial effective distances, the new effective distance being from a biological or chemical sample to the optical component, the biological or chemical samples not being the calibration target, and wherein the calibration target has a higher contrast than the biological or chemical samples, wherein the calibration target includes at least one object of known size; for each of a plurality of images acquired at the initial effective distances: using the identified optimal focus setting of the optical component to determine a correlation of the known size to a number of pixels; based on the correlation of the known size to a number of pixels, determining a size of the image; and storing second data for the imaging system to determine a second functional approximation that is derived from the sizes at the initial effective distances, the second data being stored in the at least one computer readable medium that is adapted to be communicably coupled with the imaging system, wherein the second functional approximation is operable for calculating a new effective distance that is not one of the initial effective distances from a size. for each of a plurality of initial effective distances from an optical component of the imaging system to a calibration target at a sample location, identifying an initial optimal focus setting of the optical component, wherein different initial effective distances correspond to different initial optimal focus settings of the optical component; storing first data for the imaging system to determine a first functional approximation that is derived from the initial optimal focus settings of the optical component for the initial effective distances, the first data being stored in at least one computer readable medium that is adapted to be communicably coupled with the imaging system, wherein the first functional approximation is operable for calculating a new optimal focus setting of the optical component for a new effective distance that is not one of the initial effective distances, the new effective distance being from a biological or chemical sample to the optical component, the biological or chemical samples not being the calibration target, and wherein the calibration target has a higher contrast than the biological or chemical samples, wherein the calibration target includes at least one object of known size; for each of a plurality of images acquired at the initial effective distances: using the identified optimal focus setting of the optical component to determine a correlation of the known size to a number of pixels; based on the correlation of the known size to a number of pixels, determining a size of the image; and storing second data for the imaging system to determine a second functional approximation that is derived from the sizes at the initial effective distances, the second data being stored in the at least one computer readable medium that is adapted to be communicably coupled with the imaging system, wherein the second functional approximation is operable for calculating a new effective distance that is not one of the initial effective distances from a size. using the identified optimal focus setting of the optical component to determine a correlation of the known size to a number of pixels; based on the correlation of the known size to a number of pixels, determining a size of the image; and storing second data for the imaging system to determine a second functional approximation that is derived from the sizes at the initial effective distances, the second data being stored in the at least one computer readable medium that is adapted to be communicably coupled with the imaging system, wherein the second functional approximation is operable for calculating a new effective distance that is not one of the initial effective distances from a size.