An image analysis method for determining the density of target molecules in a tissue sample depicted in a fluorescence image, the method comprising: - receiving, by an image analysis system, a digital image of a slide, the slide comprising the tissue sample, a target dot and a fluoro dot, the fluoro dot comprising a known density of fluorescence-inducing molecules, the target dot comprising a known density of target molecules, the tissue sample and the target dot having been stained in the same staining procedure with the same type of fluorescence-inducing molecules as contained in the fluoro dot, the digital image comprising intensity values of the tissue sample, intensity values of the target dot and intensity values of the fluoro dot, the fluorescence-inducing molecules being adapted to directly or indirectly bind to the target molecules during the staining procedure; - receiving, by the image analysis system: - pixel intensities of the fluoro dot depicted in the image; - pixel intensities of the target dot depicted in the image; - pixel intensities of an area of the tissue sample depicted in the image; - fluorescence-inducing molecule density information indicative of the known density of the fluorescence-inducing molecules in the fluoro dot; - target molecule density information indicative of the known density of the target molecules in the target dot; - computing the density of target molecules in the area of the tissue sample as a function of the pixel intensities of the fluoro dot, the target dot, the area of the tissue sample, the fluorescence-inducing molecule density information and the target molecule density information. - receiving, by an image analysis system, a digital image of a slide, the slide comprising the tissue sample, a target dot and a fluoro dot, the fluoro dot comprising a known density of fluorescence-inducing molecules, the target dot comprising a known density of target molecules, the tissue sample and the target dot having been stained in the same staining procedure with the same type of fluorescence-inducing molecules as contained in the fluoro dot, the digital image comprising intensity values of the tissue sample, intensity values of the target dot and intensity values of the fluoro dot, the fluorescence-inducing molecules being adapted to directly or indirectly bind to the target molecules during the staining procedure; - receiving, by the image analysis system: - pixel intensities of the fluoro dot depicted in the image; - pixel intensities of the target dot depicted in the image; - pixel intensities of an area of the tissue sample depicted in the image; - fluorescence-inducing molecule density information indicative of the known density of the fluorescence-inducing molecules in the fluoro dot; - target molecule density information indicative of the known density of the target molecules in the target dot; - pixel intensities of the fluoro dot depicted in the image; - pixel intensities of the target dot depicted in the image; - pixel intensities of an area of the tissue sample depicted in the image; - fluorescence-inducing molecule density information indicative of the known density of the fluorescence-inducing molecules in the fluoro dot; - target molecule density information indicative of the known density of the target molecules in the target dot; - computing the density of target molecules in the area of the tissue sample as a function of the pixel intensities of the fluoro dot, the target dot, the area of the tissue sample, the fluorescence-inducing molecule density information and the target molecule density information.