Dual excitation fluorescence imaging has been used as a first step towards multi-wavelength excitation/emission fluorescence spectral imaging. Target cells are transformed keratinocytes, and osteosarcoma, human breast and colon cancer cells. Mitochondrial membrane potential probes, e.g. TMRM (tetramethylrhodamine methyl ester), Mitotracker Green (Molecular Probes, Inc., Eugene OR, USA) and a recently synthesized mitochondrial oxygen probe, [PRE, (1″- pyrene butyl)-2-rhodamine ester] allow dual excitation in the UV plus in the blue-green spectral regions. Also, using the natural endogenous probe NAD(P)H, preliminary results indicate mitochondrial responses to metabolic challenges (e.g. glucose addition), plus changes in mitochondrial distribution and morphology. In terms of application to biomedicine (for diagnostics, prognostics and drug trials) three parameters have been selected in addition to the natural probe NAD(P)H, i.e. vital fluorescence probing of mitochondria, lysosomes and Golgi apparatus. It is hoped that such a multiparameter approach will allow malignant cell characterization and grading. A new area being introduced is the use of similar methodology for biotechnological applications such as the study of the hydrogen-producing alga Chlamydomonas Reinhardtii, and possible agricultural applications, such as Saccharomyces yeast for oenology. Complementation by Photoacoustic Microscopy is also contemplated, to study the internal conversion component which follows the excitation by photons.
