Alteration of Tissue Marking Dyes Depends on Used Chromogen during Immunohistochemistry

Pathological biopsy protocols require tissue marking dye (TMD) for orientation. In some cases (e.g., close margin), additional immunohistochemical analyses can be necessary. Therefore, the correlation between the applied TMD during macroscopy and the examined TMD during microscopy is crucial for the correct orientation, the residual tumour status and the subsequent therapeutic regime. In this context, our group observed colour changes during routine immunohistochemistry. Tissue specimens were marked with various TMD and processed by two different methods. TMD (blue, red, black, yellow and green) obtained from three different providers (A, B and C, and Whiteout/Tipp-Ex®) were used. Immunohistochemistry was performed manually via stepwise omission of reagents to identify the colour changing mechanism. Blue colour from provider A changed during immunohistochemistry into black, when 3,3′-Diaminobenzidine-tetrahydrochloride-dihydrate (DAB) and H2O2 was applied as an immunoperoxidase-based terminal colour signal. No other applied reagents, nor tissue texture or processing showed any influence on the colour. The remaining colours from provider A and the other colours did not show any changes during immunohistochemistry. Our results demonstrate an interesting and important pitfall in routine immunohistochemistry-based diagnostics that pathologists should be aware of. Furthermore, the chemical rationale behind the observed misleading colour change is discussed.

Fluctuating environmental light limits number of surfaces visually recognizable by colour

Small changes in daylight in the environment can produce large changes in reflected light, even over short intervals of time. Do these changes limit the visual recognition of surfaces by their colour? To address this question, information-theoretic methods were used to estimate computationally the maximum number of surfaces in a sample that can be identified as the same after an interval. Scene data were taken from successive hyperspectral radiance images. With no illumination change, the average number of surfaces distinguishable by colour was of the order of 10,000. But with an illumination change, the average number still identifiable declined rapidly with change duration. In one condition, the number after two minutes was around 600, after 10 min around 200, and after an hour around 70. These limits on identification are much lower than with spectral changes in daylight. No recoding of the colour signal is likely to recover surface identity lost in this uncertain environment.

The truth is in the detail: predators attack aposematic prey with less aggression than other prey types

Aposematic organisms are often unprofitable to predators (e.g. because of defensive chemicals) which they advertise with a conspicuous signal (e.g. bright and conspicuous colour signals). Aposematism is thought to reduce predation of prey because the colour signal increases the ability of predators to learn, recognize and remember the prey’s defensive properties. The efficacy of aposematism has been extensively documented in laboratory studies, although its benefits seem to be harder to demonstrate in the field. In this study, we compared the levels of partial and overall predation among four prey types (undefended and cryptic, undefended and warning coloured, defended and cryptic, and aposematic prey). Overall, predation of warning coloured and defended (aposematic) prey was lower than the predation for cryptic and undefended prey; however, it was the same as predation of cryptic and defended prey. Moreover, aposematic prey had higher levels of partial predation (where prey was not wholly consumed by the predator) and lower attack intensities. This suggests that prey were being taste sampled, but also might be better able to survive attacks. Therefore, the benefits of aposematism may lie not only in reducing outright predation, but also in altering a predator’s post-attack behaviour, thus leading to greater escape opportunities and post-attack survival of prey. These results reinforce the importance of examining predation in more detail rather than simply examining attack rates.

Why colour is complex: Evidence that bees perceive neither brightness nor green contrast in colour signal processing

Honey bees ( Apis mellifera Linnaeus, 1758) potentially rely on a variety of visual cues when searching for flowers in the environment. Both chromatic and achromatic (brightness) components of flower signals have typically been considered simultaneously to understand how flower colours have evolved. However, it is unclear whether honey bees actually use brightness information in their colour perception. We investigated whether free-flying honey bees can process brightness cues in achromatic stimuli when presented at a large visual angle of 28° to ensure colour processing. We found that green contrast (modulation of the green receptor against the background) and brightness contrast (modulation of all three receptors against the background) did not have a significant effect on the proportion of correct choices made by bees, indicating that they did not appear to use brightness cues in a colour processing context. Our findings also reveal that, even at a small visual angle, honeybees do not reliably process single targets solely based on achromatic information, at least considering values up to 60% modulation of brightness. We discuss these findings in relation to proposed models of bee colour processing. Therefore, caution should be taken when interpreting elemental components of complex flower colours as perceived by different animals.

Fluorescence as a means of colour signal enhancement

Fluorescence is a physico-chemical energy exchange where shorter-wavelength photons are absorbed by a molecule and are re-emitted as longer-wavelength photons. It has been suggested a means of communication in several taxa including flowers, pitcher plants, corals, algae, worms, squid, spiders, stomatopods, fish, reptiles, parrots and humans. The surface or object that the pigment molecule is part of appears to glow due to its setting rather than an actual production of light, and this may enhance both signals and, in some cases, camouflage. This review examines some known uses of fluorescence, mainly in the context of visual communication in animals, the challenge being to distinguish when fluorescence is a functional feature of biological coloration or when it is a by-product of a pigment or other molecule. In general, we conclude that most observations of fluorescence lack enough evidence to suggest they are used in visually driven behaviours. This article is part of the themed issue ‘Animal coloration: production, perception, function and application’.