A method of calibrating an imaging system for analyzing a plurality of molecular species in a sample, said method comprising: acquiring a plurality of images of the sample with an image acquisition device at a plurality of different wavelengths; comparing a region of interest associated with at least one of the images acquired at one respective wavelength to a region of interest associated with at least one of the images acquired at a different wavelength; determining a magnification factor for each of a plurality of wavelengths from a reference image, wherein the magnification factor characterizes the difference in magnification between one image taken with respect to one wavelength and another image taken with respect to a different wavelength; and aligning the plurality of images such that the region of interest associated with at least one of the images acquired at one respective wavelength corresponds to the region of interest associated with the at least one of the images acquired at the different wavelength; wherein determining the magnification factor comprises: capturing a defocused image of a calibration slide in each of the plurality of wavelengths, the calibration slide comprising a lattice of a plurality of cells arranged in an alternating pattern, each cell further comprising a plurality of pixels; determining a shading of each of the plurality of pixels so as to form a mask for discriminating between a percentage of the lightest pixels and an equal percentage of the darkest pixels; determining an area for each of a plurality of cells; determining a center for each of a plurality of cells; measuring a distance between the centers of each of the plurality of cells and their neighboring cells in the same image; refining the measurements of the areas for each of the plurality of cells and the measurements of the distances between the centers of each of the plurality of cells providing the magnification factor based on the average of the measured distances. acquiring a plurality of images of the sample with an image acquisition device at a plurality of different wavelengths; comparing a region of interest associated with at least one of the images acquired at one respective wavelength to a region of interest associated with at least one of the images acquired at a different wavelength; determining a magnification factor for each of a plurality of wavelengths from a reference image, wherein the magnification factor characterizes the difference in magnification between one image taken with respect to one wavelength and another image taken with respect to a different wavelength; and aligning the plurality of images such that the region of interest associated with at least one of the images acquired at one respective wavelength corresponds to the region of interest associated with the at least one of the images acquired at the different wavelength; wherein determining the magnification factor comprises: capturing a defocused image of a calibration slide in each of the plurality of wavelengths, the calibration slide comprising a lattice of a plurality of cells arranged in an alternating pattern, each cell further comprising a plurality of pixels; determining a shading of each of the plurality of pixels so as to form a mask for discriminating between a percentage of the lightest pixels and an equal percentage of the darkest pixels; determining an area for each of a plurality of cells; determining a center for each of a plurality of cells; measuring a distance between the centers of each of the plurality of cells and their neighboring cells in the same image; refining the measurements of the areas for each of the plurality of cells and the measurements of the distances between the centers of each of the plurality of cells providing the magnification factor based on the average of the measured distances. capturing a defocused image of a calibration slide in each of the plurality of wavelengths, the calibration slide comprising a lattice of a plurality of cells arranged in an alternating pattern, each cell further comprising a plurality of pixels; determining a shading of each of the plurality of pixels so as to form a mask for discriminating between a percentage of the lightest pixels and an equal percentage of the darkest pixels; determining an area for each of a plurality of cells; determining a center for each of a plurality of cells; measuring a distance between the centers of each of the plurality of cells and their neighboring cells in the same image; refining the measurements of the areas for each of the plurality of cells and the measurements of the distances between the centers of each of the plurality of cells providing the magnification factor based on the average of the measured distances.