Quantitative fluorescence image analysis.

Abstract

So far quantitative fluorescence histochemistry has been carried out separately by two different methods, cytofluorometry for densitometry and image analysis for morphometry. We described here a method of quantitative fluorescence image analysis to realize simultaneous densitometric and morphometric measurements. This method gives complementary informations of quantity of substances of interest together with data on shape and pattern of the biological materials.DAPI staining for DNA is employed. The fluorescence images are successively taken from SIT (Silicon Intensifier Target) camera and sent to an intelligent color terminal. The data obtained from the image analyzer are compared with cytofluorometric data. Integration technique can remove random noise from the image.Brightness values of all pixels in the frame buffer or brightness of the pixels on nuclear area were summed up. Both parameters responded linearly and proportionally to fluorescence values determined by cytofluorometry within an adequate range. Nuclear DNA measurements revealed that rat hepatocytes are composed of diploid, tetraploid and octaploid subpopulations with proportional DNA contents. All cerebellar neurons showed DNA values strictly in diploid class.The conclusion is that both densitometric and morphometric informations can be obtained simultaneously by quantitative fluorescence image analysis within an adequate dynamic range. This will enable us to extend quantitative investigation on histochemical reaction and shape analysis in variety of biological systems.