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Retinal imaging spectroscopy can provide functional maps using chromophore spectra. For example, oxygen saturation maps show ischemic areas from diabetes and venous occlusions. Obtaining retinal spatial-spectral data has been difficult due to saccades and long data acquisition times (>5 s). We present a snapshot imaging spectrometer with far-reaching applicability that acquires a complete spatial-spectral image cube in approximately 3 ms from 450 to 700 nm with 50 bands, eliminating motion artifacts and pixel misregistration. Current retinal spectral imaging approaches are incapable of true snapshot operation over a wide spectral range with a large number of spectral bands. Coupled to a fundus camera, the instrument returns true color retinal images for comparison to standard fundus images and for image validation while the patient is still dilated. Oxygen saturation maps were obtained with a three-wavelength algorithm: for healthy subjects arteries were approximately 95% and veins 30 to 35% less. The instrument is now undergoing clinical trials.

Abstract. The imaging of living cells and tissues using laser-scanning microscopy is offering dramatic insights into the spatial and temporal controls of biological processes. The availability of genetically encoded labels such as green fluorescent protein (GFP) offers unique opportunities by which to trace cell movements, cell signaling or gene expression dynamically in developing embryos. Two-photon laser scanning microscopy (TPLSM) is ideally suited to imaging cells in vivo due to its deeper tissue penetration and reduced phototoxicity; however, in TPLSM the excitation and emission spectra of GFP and its color variants [e.g., CyanFP (CFP); yellowFP (YFP)] are insufficiently distinct to be uniquely imaged by conventional means. To surmount such difficulties, we have combined the technologies of TPLSM and imaging spectroscopy to unambiguously identify CFP, GFP, YFP, and redFP (RFP) as well as conventional dyes, and have tested the approach in cell lines. In our approach, a liquid crystal tunable filter was used to collect the emission spectrum of each pixel within the TPLSM image. Based on the fluorescent emission spectra, supervised classification and linear unmixing analysis algorithms were used to identify the nature and relative amounts of the fluorescent proteins expressed in the cells. In a most extreme case, we have used the approach to separate GFP and fluorescein, separated by only 7 nm, and appear somewhat indistinguishable by conventional techniques. This approach offers the needed ability to concurrently image multiple colored, spectrally overlapping marker proteins within living cells.

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tions due to the self-energy, vacuum polarization, magnetic interaction, and retardation. Nearly all of these corrections have been calculated by expansions in terms of the parameter Za, but experiments are already being conducted in the region where Za > 1. This is a great challenge to the theory. An approach to ab initio relativistic many-electron molecular Hartree-Fock calculations is under way.