Increased depth of field and stereo pairs of fluorescence micrographs via inverse filtering and maximum‐likelihood estimation

Image processing methods are presented for effectively increasing the depth of field and for generating stereo pairs of fluorescence micrographs from a conventional optical microscope. In developing these methods the slice theorem of computed tomography is used. In this way the image reconstruction problem is reduced to one of processing only two‐dimensional arrays rather than three‐dimensional arrays and the classical difficult problem of restoring missing Fourier components within the missing cone region is circumvented. Two different approaches to such processing are presented. One approach is based on inverse filtering. Another approach is based on previous development of iterative image restoration algorithms for quantum‐limited incoherent imagery, founded on maximum‐likelihood estimation. Limited experimentation with real micrographs shows that both approaches work well. Some preliminary comparisons are made between the different variations of the methods tested, which point out the advantages and present limitations. Both methods can be implemented on IBM‐AT‐compatible computers with relatively fast execution times. Advantages that these methods have over confocal microscopy are (i) the optical and computing equipment required is less expensive, and (ii) a conventional microscope when set up properly can have much better light sensitivity than a confocal microscope.