Copper-Metallothionein Autofluorescence† (2009)

We tend to curse background autofluorescence when looking at immunofluorescence preparations from liver tissue, but we may need to reconsider. Intracytoplasmic accumulation of copper and/or copper-associated protein deposition characterizes Wilson disease, copper toxicosis, and more commonly, chronic cholestasis. We have examined unstained 6-μm-thick frozen sections from snap-frozen samples of livers removed at transplantation for Wilson disease or biliary disease. We used a Leica LMD6000 microscope equipped with a Leica EL6000 compact light source and a BGR (blue-green-red) filter in the blue position (Excitation filter 420/30, dichromatic mirror 415, suppression filter 465/20, as supplied by Leica Microsystems Ltd., Milton Keynes, UK). Hepatocytes in cases of Wilson disease contained bright red-orange granules (Fig. 1A) that were not evident with the red or green filter (not shown). The same frozen sections were later stained for rhodanine and orcein using a standard routine histochemical method. Using the repositioning memory software available on the LMD6000, the same spots were relocalized and rephotographed. Both rhodanine (Fig. 1B) and orcein stains demonstrate copper and/or copper-associated protein granules which colocalized with the fluorescent granules (examples shown by matching arrows, arrowheads, and asterisks). Figures 1C and 1D show that bright red-orange granules in unstained frozen sections colocalized with orcein-positive granules in periseptal hepatocytes of a liver removed at transplantation for primary biliary cirrhosis. We believe our findings are consistent with previous work by Okabe et al.1 and Stillman et al.2 According to Okabe et al.,1 copper-metallothionein autofluorescence is due to the presence of Copper-Thiolate complexes within metallothionein. Both orcein and rhodanine stains or just paraffin embedding quenched the autofluorescence signal, possibly due to dissociation of the protein-copper complexes3 or another physicochemical mechanism.2 Copper-metallothionein autofluorescence was not observed in samples of normal liver. NAD(P)H, reduced nicotinamide adenine dinucleotide phosphate. Conventional bright-field microscopy is based on the observer's perception of colors derived from the interactions between light wavelengths and dyes specific for certain tissue/cell structures (e.g., hematoxylin and nucleus). Fluorescence microscopy is based on the interactions between light wavelengths, molecules, and filters/dichromatic mirrors, which may unmask specific combinations for the identification of particular structures, in this case Copper-Thiolate complexes. Fluorescence is “a beautiful manifestation of the interaction of light with matter”.4 Autofluorescence and its potential applications deserve more consideration in liver microscopy and diagnostic liver pathology.5 Autofluorescence allows microdissection of tissue sections by avoiding the use of fixatives and dyes which may interfere with downstream molecular work. The possible use of autofluorescence for tissue copper quantitation needs to be explored. Other endogenous fluorophores of potential interest include lipopigments, matrix proteins such as collagen and elastin, NADPH (reduced nicotinamide adenine dinucleotide phosphate) and flavins, drugs, bile constituents, vitamin A, and porphyrins. Autofluorescence-based techniques have been used for the assessment of fibrosis, cell metabolism, and differentiation, discrimination between neoplastic and non-neoplastic tissue, and studies of microorganisms and drug interactions.5 The authors wish to thank Ms Anne Rayner and Mr Chris Starling for the histochemical stains.