Techniques for reducing the interfering effects of autofluorescence in fluorescence microscopy: improved detection of sulphorhodamine B-labelled albumin in arterial tissue.
Measurements of the transport of circulating sulphorhodamine B-labelled albumin into the arterial wall, made by applying digital imaging fluorescence microscopy to sections of arteries fixed in situ, are limited in sensitivity by the low levels of tracer fluorescence and high levels of autofluorescence emitted from the tissue. Three attempts to improve these ratios are described. In the first, spectra of the tracer in solution and of arterial autofluorescence were used to design novel microscope filters for rhodamine-like dyes. By exciting with the rarely used yellow lines of the mercury arc lamp and detecting a narrow band of emission with Stokes shifts as small as 15 nm, the ratio of tracer fluorescence to autofluorescence was tripled. In the second, effects of different fixatives were investigated. Using a model system, it was confirmed that Karnovsky's fixative gives good tracer immobilization but elevates autofluorescence, whereas fixative-free buffer solutions give low autofluorescence but do not retain the tracer. It was further found that simple formaldehyde-based fixatives, hitherto considered to be poor fixatives of albumin, immobilized the tracer as well as the glutaraldehyde-based fixative, whilst giving autofluorescence levels comparable to those seen with buffer alone; they therefore give excellent tracer fluorescence to autofluorescence ratios. In the third, lowering specimen temperature by 50 degrees C was found to increase the intensity of tracer fluorescence by 30% whilst autofluorescence was unaffected. These data may have relevance to microscopical studies using other tissues and fluorescent tracers.
Staughton
T J
TJ
School of Animal and Microbial Sciences, University of Reading, Whiteknights P O Box 228, Reading RG6 6AJ, UK.
McGillicuddy
C J
CJ
Weinberg
P D
PD
D000818:Animals; D001158:Arteries / Q000378:metabolism*; D005374:Filtration; D005456:Fluorescent Dyes; D005557:Formaldehyde; D007089:Image Enhancement / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods*; D011817:Rabbits; D012235:Rhodamines; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000378:metabolism*; D013048:Specimen Handling; D013696:Temperature
D005456:Fluorescent Dyes; D012235:Rhodamines; D005557:Formaldehyde; C022027:lissamine rhodamine B; D012710:Serum Albumin, Bovine