Fluorescence intensity in a spectrofluorometer is proportional to the concentration of analyte over a wide dynamic range and is, therefore, an excellent quantitative analytical tool. In this paper we use the techniques of deconvolution and convolution to extend the utility of quantitative fluorescence to the measurement of analyte concentrations inside cells and their organelles. This permits us to assess the physiological state of living cells in situ.
To successfully turn a voxel within a 3D image into a microcuvette, 2 conditions must be met. First, to be sure the voxel is associated with only one fluorescent structure, the object that is being measured must be fully resolved from neighboring objects in all three dimensions. For structures separated by distances larger than about 300nm in the xy plane or 600nm in z (where z is the optical axis), this can be achieved with either digital deconvolution of 3D widefield images or by confocal microscopy.