A method of focusing, by a high content imaging system, a biological sample positioned on a plate (116, 152), the plate (116, 152) having inner and outer surfaces, the high content imaging system having an imaging component and a camera for imaging biological samples, the outer surface of the plate, the inner surface of the plate, and the biological sample being positioned in this order along a z-axis, the method comprising: shining a laser light beam through the imaging component toward the plate (116, 152) to cause reflections of the laser light from the inner and outer surfaces of the plate (116, 152) to pass back through the imaging component and to the camera; producing, by the camera, an image of the received reflections from the inner and outer surfaces of the plate (116, 152); processing the image by a computerized device to estimate a z-distance between the imaging component and the inner surface of the plate (116, 152); and moving the imaging component to a z-position at which the biological sample is focused, based on the estimated z-distance, and wherein processing the image comprises: cropping the image to obtain a desired region, determining representative values for each column of the desired region, wherein each row/column position corresponds to a single pixel of the image and storing the values in a linear array, determining a local minimum of the linear array; determining a threshold value based on the local minimum; identifying and differentiating at least two objects from the linear array, the at least two objects respectively corresponding to the received reflections from the inner and outer surfaces of the plate (116, 152); determining a column position of the received reflection of the inner surface of the plate (116, 152) based on the identified and differentiated objects; and estimating the z-distance based on the column position determined for the inner surface reflection of the plate (116, 152). shining a laser light beam through the imaging component toward the plate (116, 152) to cause reflections of the laser light from the inner and outer surfaces of the plate (116, 152) to pass back through the imaging component and to the camera; producing, by the camera, an image of the received reflections from the inner and outer surfaces of the plate (116, 152); processing the image by a computerized device to estimate a z-distance between the imaging component and the inner surface of the plate (116, 152); and moving the imaging component to a z-position at which the biological sample is focused, based on the estimated z-distance, and wherein processing the image comprises: cropping the image to obtain a desired region, determining representative values for each column of the desired region, wherein each row/column position corresponds to a single pixel of the image and storing the values in a linear array, determining a local minimum of the linear array; determining a threshold value based on the local minimum; identifying and differentiating at least two objects from the linear array, the at least two objects respectively corresponding to the received reflections from the inner and outer surfaces of the plate (116, 152); determining a column position of the received reflection of the inner surface of the plate (116, 152) based on the identified and differentiated objects; and estimating the z-distance based on the column position determined for the inner surface reflection of the plate (116, 152). cropping the image to obtain a desired region, determining representative values for each column of the desired region, wherein each row/column position corresponds to a single pixel of the image and storing the values in a linear array, determining a local minimum of the linear array; determining a threshold value based on the local minimum; identifying and differentiating at least two objects from the linear array, the at least two objects respectively corresponding to the received reflections from the inner and outer surfaces of the plate (116, 152); determining a column position of the received reflection of the inner surface of the plate (116, 152) based on the identified and differentiated objects; and estimating the z-distance based on the column position determined for the inner surface reflection of the plate (116, 152).