Techniques for reducing the interfering effects of autofluorescence in fluorescence microscopy: improved detection of sulphorhodamine B‐labelled albumin in arterial tissue

Abstract

SummaryMeasurements 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 8C 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.

Modern Trends in Imaging X: Spectral Imaging in Preclinical Research and Clinical Pathology

“…However, quantum-dot-based labels have not yet achieved widespread adoption, in part due to their own properties as well as to other challenges that fluorescence-based methods face in general [38]. One of these is interference from various sources of autofluorescence, especially common with formalin-fixed tissue samples [40][41][42][43][44][45]. Additional hurdles include the requirement for complex and expensive microscopes, and interference with typical pathology workflow.…”

“…Hard-to-resolve mixtures of fluorescent signals in many or most separate channels can be inevitable when autofluorescence is present. Autofluorescence is the often-unwanted spontaneous fluorescence emission of the cells or tissues being imaged not arising from the exogenous dyes used for labeling [44,45].…”

Efficient simulation of autofluorescence effects in microscopic lenses

“…Common sources of optical noise include unwanted emission by autofluorescent cells or tissues, emission of fluorophores which are not bound to target of interest and autofluorescence produced by a microscope itself. Several methods can be employed during measurement to reduce autofluorescence which originates from the sample [5][6][7][8] . There are theoretical models able to predict fluorescence from the sample 9 .…”

Time‐Gated Luminescence Microscopy

“…There are instances where the detection of rare-event signals using conventional fluorescence techniques is exceedingly difficult (or impossible), and consequently, where TGL microscopy has greatest utility. [17][18][19][20] Luminescent probes based on the lanthanides Eu 3+ and Tb 3+ were described in the 1960s, but effective immunofluorophores using these compounds were not FIGURE 1. Pulse fluorometry or time-gated luminescence techniques operate on timescales orders of magnitude greater than frequency domain methods.…”