Rapid Color Change Research Articles

Carbazole-Anthranyl π-Conjugates as Small and Stable Aggregation-Induced Emission-Active Fluorogens: Serving as a Reusable and Efficient Platform for Anticounterfeiting Applications with an Acid Key and Multicolor Ink for a Quill Pen.

Being a unique, simple, and inexpensive approach, continuous development on the fluorescence-based technologies remains active in fluorescent anticounterfeiting. A number of polymeric, nano-, carbon dot, and rare-earth oxide materials were preferably explored for such applications, but the complex synthesis, purity, and high cost are the major concerns to make these materials accessible for commercial applications. To address these difficulties, we herein report simple mono-carbazole-linked anthranyl π-conjugates that are synthesized in a gram scale via an inexpensive and convenient route. These unsymmetrically substituted new π-conjugates are found to be promising blue-shifted aggregation-induced emission-active fluorogens (AIEgens) having a distinct color on varying substituents with electron-rich (−NEt2) and electron-poor (−CN) functionalities. The direct link of a single carbazole unit with an anthracenyl π-conjugate possibly enforces the achievement of a highly twisted molecular structure, accountable for the AIE characteristics. The π-conjugate with −NEt2 substituents is established to be highly sensitive under protonation–deprotonation stimuli by a sharp and rapid fluorescence color change [yellow (Φf = 37%) to green (Φf = 39.4%)] in the solid state (no fluorescence on/off). Upon the exposure of the base vapors (deprotonation), the original emission color (yellow) comes back. Such reversible and also repeatable acidchromism is demonstrated to be perfectly suitable for anticounterfeiting applications by marking the AIEgen on the paper that shows the bright image of the AIEgen under the UV torch (365 nm). Almost equal efficiencies by fabricating on different surfaces such as polythene paper and a fresh leaf are observed. While these spots can be duplicated with typical yellow fluorescent dyes, our AIEgen can easily be differentiated with the acid key. The emission color change of this AIEgen from yellow to green under acid stimuli is distinctly defined compared to other dyes and vividly recognized by naked eyes. Thus, one can combat the counterfeiters with the acid key. The reversible color-changing behavior on the paper remains intact even after six consecutive days of exposure to sunlight, and the AIEgen is thermally stable up to 445 °C. Further, this compound is also utilized as ink (10 μM 1,4-dioxane solution) where a pigeon feather is used as a quill pen. The mechanistic insights behind these facts have also been proposed and validated wherever possible.

Microbubbles on kidney stones contribute to the twinkling artifact in humans

The color Doppler ultrasound twinkling artifact, a rapid color change that highlights kidney stones, was recently attributed to surface crevice microbubbles on ex vivo stones because twinkling was affected by changes in hydrostatic pressure. However, it was unclear whether crevice bubbles existed on in situ human kidney stones, and, if so, how smooth-surfaced stones could harbor the crevice bubbles that give rise to twinkling. Here, 8 human subjects with known kidney stones were exposed to 4 atmospheres absolute (ATA) while breathing air inside a hyperbaric chamber; twinkling was monitored and quantified with a research ultrasound system. At 3 and 4 ATA, twinkling was significantly reduced by an average of 35% and 39%, respectively (p = 0.04). Then, ex vivo kidney stones that twinkled were exposed to micro-computed tomography (μCT) and hypobaric pressures while submerged in water. Regions of low x-ray attenuation within the stone (i.e., microcracks) at 1 ATA expanded when the pressure was reduced to 0.1 ATA. These results support the theory that microbubbles are present on kidney stones in the human body and that microbubbles may be internal as well as external to the kidney stone. [Work supported by NSBRI through NASA NCC 9-58 and NIH DK043881.]

Incorporation of 1,3-Free-2,6-Connected Azulene Units into the Backbone of Conjugated Polymers: Improving Proton Responsiveness and Electrical Conductivity.

Azulene as a potential building block for constructing organic/polymeric conjugated materials has attracted more and more attention due to its unique chemical structure and physicochemical properties. However, up to now, most reported azulene-based conjugated polymers have been dominated by the connection of the five-membered ring of azulene through 1,3-positions. Herein, by incorporating 1,3-free-2,6-connected azulene units into the polymeric backbone, two azulene-based all-carbon conjugated polymers P1 and P2 with different connection ways of 2,6-azulene and 2,7-fluorene units were presented. Protonation of these two polymers with trifluoroacetic acid leads to rapid and reversible color changes in both the solution and thin-film state. Moreover, these 1,3-free-2,6-connected azulene-based conjugated polymers exhibit high electrical conductivity (2.94 and 0.32 S/cm for P1 and P2, respectively) in thin film when doped by trifluoromethanesulfonic acid.

Social and physiological drivers of rapid colour change in a tropical toad

Dynamic sexual dichromatism occurs when males and females differ in colouration for a limited time. Although this trait has been primarily studied in cephalopods, chameleons, and fishes, recent analyses suggest that dynamic dichromatism is prevalent among anurans and may be mediated through sexual selection and sex recognition. Yellow toads, Incilius luetkenii, exhibit dynamic dichromatism during explosive breeding events at the onset of the rainy season: males change from a cryptic brown to a bright yellow and back again during the brief mating event. We tested the hypothesis that dynamic dichromatism in yellow toads is influenced by conspecific interactions and mediated through sex hormones and stress hormones. We placed male toads into one of four social treatments (with three other males, one male, one female, or no other toads). Immediately before and after each one-hour treatment, we quantified male colour with a reflectance spectrometer and we collected a blood sample to assess plasma concentrations of both testosterone and corticosterone. We found that males held with conspecific animals showed the brightest yellow colour and showed little or no change in their corticosterone levels. Across treatments, toads with duller yellow colour had higher levels of corticosterone. Male colour showed no association with testosterone. Interestingly, males showed substantial temporal variation in colour and corticosterone: toads were duller yellow and exhibited greater levels of corticosterone post-treatment across subsequent days at the onset of the rainy season. Our findings reveal that both conspecific interactions and corticosterone are involved in the dynamic colour change of yellow toads.

Nano molar level chromogenic and fluorogenic sensing of heavy metal ions using multi-responsive novel Schiff base as a dual mode chemosensor

A Schiff base centered chemosensor namely (2E,NE)-N-(2,4-dihydroxybenzylidene)-2-(quinolin-2-ylmethylene)hydrazinecarbothioamide i.e.SB has been synthesized and further utilized for the selective and sensitive detection of four metal ions through dual channels based on absorption and fluorescence properties. The distinctive recognition of Ni2+, Cu2+, Co2+, and Cd2+ in an aqueous solution was achieved by observation with the naked eye rapid color change, analyzing the absorption and fluorescence spectrum of chemosensor SB in the absence and presence of specific metal ion at respective concentration. The synthesized chemosensor SB offered extremely lower detection limit of nano molar level employing either the absorption or fluorescence method. The strong binding constant values such as 9.02 × 105 M−1 for Ni2+, 11.21 × 105 M−1 for Cu2+, 7.52 × 105 M−1 for Co2+ and 6.73 × 105 M−1 for Cd2+ indicate strong complexation between chemosensor SB and respective metal ion. The mechanism for change in the absorption and fluorescence properties of SB involves the interaction of respective metal ion with sulfur and oxygen functionality in SB molecule followed by the formation of ground state non-fluorescent complex via static quenching process. The TD-DFT studies for the theoretical calculations are in support of experimental interpretations. Pleasingly, the present approach allows quantitative determination of said metal ions in water samples at nano molar level.

Tactical Tentacles: New Insights on the Processes of Sexual Selection Among the Cephalopoda.

The cephalopods (Mollusca: Cephalopoda) are an exceptional class among the invertebrates, characterised by the advanced development of their conditional learning abilities, long-term memories, capacity for rapid colour change and extremely adaptable hydrostatic skeletons. These traits enable cephalopods to occupy diverse marine ecological niches, become successful predators, employ sophisticated predator avoidance behaviours and have complex intraspecific interactions. Where studied, observations of cephalopod mating systems have revealed detailed insights to the life histories and behavioural ecologies of these animals. The reproductive biology of cephalopods is typified by high levels of both male and female promiscuity, alternative mating tactics, long-term sperm storage prior to spawning, and the capacity for intricate visual displays and/or use of a distinct sensory ecology. This review summarises the current understanding of cephalopod reproductive biology, and where investigated, how both pre-copulatory behaviours and post-copulatory fertilisation patterns can influence the processes of sexual selection. Overall, it is concluded that sperm competition and possibly cryptic female choice are likely to be critical determinants of which individuals' alleles get transferred to subsequent generations in cephalopod mating systems. Additionally, it is emphasised that the optimisation of offspring quality and/or fertilisation bias to genetically compatible males are necessary drivers for the proliferation of polyandry observed among cephalopods, and potential methods for testing these hypotheses are proposed within the conclusion of this review. Further gaps within the current knowledge of how sexual selection operates in this group are also highlighted, in the hopes of prompting new directions for research of the distinctive mating systems in this unique lineage.

Adaptive colour change and background choice behaviour in peppered moth caterpillars is mediated by extraocular photoreception

Light sensing by tissues distinct from the eye occurs in diverse animal groups, enabling circadian control and phototactic behaviour. Extraocular photoreceptors may also facilitate rapid colour change in cephalopods and lizards, but little is known about the sensory system that mediates slow colour change in arthropods. We previously reported that slow colour change in twig-mimicking caterpillars of the peppered moth (Biston betularia) is a response to achromatic and chromatic visual cues. Here we show that the perception of these cues, and the resulting phenotypic responses, does not require ocular vision. Caterpillars with completely obscured ocelli remained capable of enhancing their crypsis by changing colour and choosing to rest on colour-matching twigs. A suite of visual genes, expressed across the larval integument, likely plays a key role in the mechanism. To our knowledge, this is the first evidence that extraocular colour sensing can mediate pigment-based colour change and behaviour in an arthropod.

Novel near-infrared pH-sensitive cyanine-based fluorescent probes for intracellular pH monitoring

Real-time monitoring of intracellular pH fluctuation is of great significance for diagnosis of diseases and study of related physiological and pathological processes. A novel near-infrared (NIR) pH-activated parent probe 4 was developed, and further functionalized to afford probes 6 and 7. With the decrease of pH from 11 to 2, they all exhibited a new absorption peak and strong fluorescence emission in NIR region, accompanied by rapid solution color change from pink to pale green. Moreover, the probes’ fluorescence responses toward pH fluctuations were reversible and stable, and the pseudo-linear relationship between the fluorescence intensity and pH value can be used to detect pH value in 2.76–6.45 range quantitatively. Finally, probe 6 and 7 were successfully applied to monitor the fluctuation of intracellular pH by a fluorescence turn-on mode, and the imaging results confirmed their low cytotoxicity and satisfactory cell-membrane permeability, as well as their application prospect in disease diagnosis (such as tumor specific imaging) or assist in study of pH-related biological processes in wide acidic environment.

Observation on rapid physiological color change in Giant tree frog Rhacophorus smaragdinus (Blyth, 1852) from Namdapha Tiger Reserve, Arunachal Pradesh, India

Many poikilotherms have the ability to change body color for homeostatic regulation, conspecific communication or predator deterrence. Physiological color change is a rapid, reversible mode of color change regulated by neuromuscular or neuroendocrine system and has been observed in several anuran species. Here we report the occurrence of physiological color change in the tree frog Rhacophorussmaragdinus (Blyth, 1852) (Amphibia, Anura, Rhacophoridae) for the first time from Namdapha Tiger Reserve, Arunachal Pradesh, India. Probable proximate causes of the behavior are discussed along with an overview of physiological color change in species of the family Rhacophoridae and nature of color change observed.

A highly selective and sensitive turn on optical probe as a promising molecular platform for rapid detection of Zn (II) ion in acetonitrile medium: Experimental and theoretical investigations

A highly Zn2+ ion responsive novel cation sensor (L) based on the dual mechanism (ESIPT/ICT) was designed, synthesized and characterized. The sensor L shows great turn on selectivity for Zn2+ ion in 1:1 stoichiometric manner via rapid color change and 5-fold fluorescence enhancement in CH3CN over other interfering cations. The addition of Zn2+ ion to L, triggers the deprotonation of imidolic and hydroxylic protons of L, which in turn shows an enhanced red shift in both UV–Vis and fluorescence spectra of the sensor. The limit of detection and binding constant obtained are well within those reported for Zn2+ ion. The theoretical calculations were correlated with experimental results to understand the sensing mechanism between host guest interactions and L distinguishes Zn2+ from Cd2+. Furthermore, bioimaging studies were undertaken to investigate practical usefulness of L to recognize Zn2+ ion using HeLa cells.

Rapidly Visualizing the Membrane Affinity of Gene Vectors Using Polydiacetylene-Based Allochroic Vesicles.

The high-throughput screening of chemically active substances has aroused widespread interest in recent years. The screening of drug carriers is usually ignored, although they interact directly with physiological barriers and target cells, and they determine the fate and efficacy of drugs in vivo. In this work, a series of polydiacetylene (PDA) vesicles (ca. 550 nm) that simulate the cell membrane are constructed to detect the membrane affinity of gene vectors. The surface potentials of vesicles are adjusted by changing the phospholipid composition using different charged compounds. All vesicles show the rapid color changes upon the addition of gene vectors by the naked eye within <5 min. The optimized 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC)-PDA vesicles display the most sensitive discoloration response to the commercially available gene vectors, including Lipofectamine 2000, Entranster-H4000, and polyethylenimine. The logarithm of transfection efficiency for green fluorescent protein plasmid (pGFP) mediated by these three vectors in L02 and HepG2 cells demonstrate an excellent linear correlation with the logarithm of membrane affinity (log Kb) of the gene vectors detected by DMPC-PDA vesicles. This rapid visualization method not only allows the in vitro membrane affinity prediction of gene vectors that greatly contributes to the gene transfection efficiency, but also offers a universal strategy for the potential high-throughput screening of various carrier materials featuring high cell affinity.

Etiology of the color Doppler twinkling artifact on kidney stones

Rapid Doppler shifts highlight some kidney stones with a rapid change in color in ultrasound imaging which is called the “twinkling artifact.” Many hypotheses exist to describe the mechanism of twinkling, including the hypothesis that microbubbles are stabilized in crevices on the surface of kidney stones. The objective is to evaluate the distribution of stable microbubbles on kidney stone surfaces with humidity-controlled scanning electron microscopy (ESEM) and determine whether any sub-surface characteristics as assessed with micro-computed tomography (µCT) may contribute to twinkling. A Verasonics® research ultrasound system with the Verasonics L22-14 and Philips/ATL L7-4 and C5-2 transducers quantified twinkling. Results show a correlation between voids within kidney stones and the locations of twinkling on the stone surface. These voids had a calculated free-field Minneart resonant frequency of 3–9 MHz depending on the size. ESEM revealed that surfaces that twinkled had more crevices with radii corresponding to bubbles with a free-field resonant frequency of 3–7 MHz. These results suggest that internal voids and surface crevices contribute to the twinkling artifact on kidney stones.

Comparative transcriptome analysis of genes involved in anthocyanin biosynthesis in the pink-white and red fruits of Chinese bayberry (Morella rubra)

Chinese bayberry (Morella rubra Sieb. and Zucc.) is an ideal fruits crop for fruits color research because of the rapid color changes that occur during ripening. Some structural genes and transcription factors involved in the anthocyanin metabolic pathway have been found in the red fruits variety of Chinese bayberry, but the mechanisms of pink-white color formation in Chinese bayberry fruits have not been reported. The red fruits of the Dongkui (DK) cultivar and the pink-white fruits of the Baidongkui (BDK) strain of Chinese bayberry were selected for transcriptome analysis. A total of 119,701 unigenes were obtained, of which 41.43% were annotated in the non-redundant (Nr) protein database. The total number of differentially expressed genes (DEGs) between the breaker fruits (BDK2) and young fruits (BDK1) was 9024, with 4993 upregulated, including DFR1, DFR2, UFGT and MYB1. The total number of DEGs between the ripe fruits (BDK3) and the breaker fruits (BDK2) was 5226, of which 2592 genes were upregulated. We found enhanced expression of structural genes, such as CHS, CHI, F3H, DFR1, DFR2, F3’H, ANS, UFGT and transcription factors such as MYB1, bHLH1. The total number of DEGs between the ripe fruits of DK (DK3) and BDK (BDK3) was only 630, of which 345 genes were upregulated. We observed enhanced expression of CHS, F3H, DFR1, DFR2, F3’H (88.3%), ANS (71.5%), and UFGT (93.0%). Hierarchy cluster analysis showed that DK3 berries were closest to BDK3 berries and furthest from BDK1 berries based on gene expression. We confirmed the results of our transcriptome analysis using quantitative real-time PCR, anthocyanin content analysis, and measurement of CIRG. Together, our results showed that the BDK pink-white fruits were formed by the upregulated expression of anthocyanin biosynthetic genes and regulatory factors. However, the reduced expression of key anthocyanin biosynthetic genes at the late stage of fruits development in BDK3 fruits compared with DK3 fruits resulted in the failure to form red fruits. Our findings shed light on the regulatory mechanisms and metabolic processes during pink-white fruits ripening of Chinese bayberry.

Label-free colorimetric nanosensor with improved sensitivity for Pb2 + in water by using a truncated 8–17 DNAzyme

Water pollution accidents, such as the Flint water crisis in the United States, caused by lead contamination have raised concern on the safety of drinking water distribution systems. Thus, the routine monitoring of lead in water is highly required and demands efficient, sensitive, cost-effective, and reliable lead detection methods. This study reports a label-free colorimetric nanosensor that uses unmodified gold nanoparticles (AuNPs) as indicators to enable rapid and ultra-sensitive detection of lead in environmental water. The 8–17 DNAzyme was truncated in this study to facilitate the detachment of single-stranded DNA fragments after substrate cleavage in the presence of Pb2+. The detached fragments were adsorbed over AuNPs and protected against salt concentration-induced aggregation. Accordingly, high Pb2+ would result in rapid color change from blue to pink. The established sensing principle achieved a sensitive limit of detection of 0.2×10–9 mol/L Pb2+, with a linear working range of two orders of magnitude from 0.5×10–9 mol/L to 5×10–9 mol/L. The selectivity of the nanosensor was demonstrated by evaluating the interfering metal ions. The developed nanosensor can serve as a substitute for the rapid analysis and monitoring of trace lead levels under the drinking water distribution system and even other environmental water samples.

Colorimetric and turn-on Fluorescence Chemosensor for Hg2+ Ion Detection in Aqueous Media.

A new rhodamine 6G based fluorescent and colorimetric chemosensor, containing N-methyl imidazole nucleus, for the selective detection of Hg2+ ion was designed and synthesized. The results of UV-Vis and fluorescence spectral study indicated that the receptor is selective and sensitive towards Hg2+ with no noticeable interference with other competitive metal ions. The addition of Hg2+ to the receptor induced a rapid color change to pink from colorless and the turn-on fluorescence response toward Hg2+ among different cations was studied. The stoichiometric ratio of 1:1 between the receptor and Hg2+ was supported by Job's plot. The color change and turn-on fluorescence response upon addition of Hg2+ ion was ascribed by the spirolactam ring-opening mechanism. The probable mode of binding between the receptor and Hg2+ was confirmed by 1H NMR and Mass spectral study. For the practical application, its electrospun nanofiber test strips successfully applied to recognize Hg2+ ion in aqueous media. Graphical Abstract Schematic representation of Hg2+ detection by rhodamine based sensor.

A simple functionalized silica microsphere for fast PETN vapor detection based on fluorescence color changes via a catalyzed oxidation process.

In this work, we report a new fluorescent material for pentaerythritol tetranitrate (PETN) vapor detection. A series of fluorene substituted vinyl-SiO microspheres with different ratios has been designed and easily synthesized. Sensing films on quartz plates were obtained by a solid phase transfer method. With a specific diameter of SiO and a specific density of surrounding fluorene, the microspheres performed a rapid fluorescence color change via oxidation with highly selective PETN catalysis. The emission peak at 355 nm was rapidly quenched while an enhancement at 525 nm appeared under exposure to PETN saturated vapor. Herein, we present this fluorescent silica nanoparticle as an excellent sensing material towards vapor phase PETN, making contributions to many fields such as public security and military use.

Two novel colorimetric probes (5-HMBA-FH and 3-HMBA-FH) based on fluorescein for copper(II) ion detection

Two novel isomeric colorimetric probes are established for simultaneous determination of copper ions using 2-hydroxy-5-methoxybenzaldehyde fluorescein hydrazone (5-HMBA-FH) and 2-hydroxy-3-methoxybenzaldehyde fluorescein hydrazone (3-HMBA-FH). They are synthesized by reacting fluorescein hydrazide with 2-hydroxy-5-methoxybenzaldehyde and 2-hydroxy-3-methoxybenzaldehyde, respectively, and then characterized by 1H-NMR, 13C-NMR, and infrared spectrum. The addition of copper ions to the solutions of two novel colorimetric probes can generate the obviously peaks at 498 nm in UV–vis absorption spectra along with a rapid colour change from colourless to dark yellow. The detection limits of the method for Cu2+ ion were 3.442 × 10−6 mol/L and 3.682 × 10−6 mol/L separately for 5-HMBA-FH and 3-HMBA-FH, respectively. The additions of other metal ions hardly affect the determination of copper ions. The proposed method was successfully applied to the analysis of Cu2+ ions in various samples. This method possesses high sensitivity, high simplicity, and minimized interference and will provide a great advantage in detecting copper ions in the environment, food, and medical applications.

Rapid colour changes in Euglena sanguinea (Euglenophyceae) caused by internal lipid globule migration

ABSTRACTThe accumulation of red pigments, frequently carotenoids, under chronic stress is a response observed in diverse kinds of eukaryotic photoautotrophs. It is thought that red pigments protect the chlorophyll located underneath by a light-shielding mechanism. However, the synthesis or degradation of carotenoids is a slow process and this response is usually only observed when the stress is maintained over long periods of time. In contrast, rapid colour changes have been reported in the euglenophyte Euglena sanguinea. Here we study the ecophysiological process behind this phenomenon through chlorophyll fluorescence, and pigment, colour and ultrastructural analyses. Reddening in E. sanguinea was due to the presence of a large amount of free and esterified astaxanthin (representing 80% of the carotenoid pool). The process was highly dynamic, shifting from green to red in 8 min (and vice-versa in 20 min). This change was not due to de novo carotenogenesis, but to the relocation of cytoplasmic lipid globules where astaxanthin accumulates. Thus, red globules were observed to migrate from the centre of the cell to peripheral locations when exposed to high light. Globule migration seems to be so efficient that other classical photoprotective mechanisms are not operative in this species. Despite the presence and operation of the diadino-diatoxanthin cycle, non-photochemical quenching was almost undetectable. Since E. sanguinea forms extensive floating colonies, reddening can be observed at a much greater scale than at the cellular level and the mechanism described here is one of the fastest and most dramatic colour changes attributable to photosynthetic organisms at cell and landscape level. In conclusion E. sanguinea shows an extremely dynamic and efficient photoprotective mechanism, based more on organelle migration than on carotenoid biosynthesis, which prevents excess light absorption by chlorophylls reducing the need for other protective processes related to energy dissipation.

A green colorimetric approach towards detection of arsenic (III): A pervasive environmental pollutant

The invasion of arsenic is slowly rising in aqueous medium which necessitates a rapid and cost effective detection. In quest to explore an effective way of monitoring arsenic (III) in aqueous medium, we report here a colorimetric method with novel green receptor viz. glucose in gold nanoparticles platform. With an average size of ∼44 nm and plasmon peak at ∼520 nm, these glucose functionalized gold nanoparticles show excellent selectivity towards arsenic (III) with a rapid color change from red to bluish. The detection unit is found to possess linearity in the range of 1–14 ppb with a remarkable limit of detection of 0.53 ppb which is well below the WHO guided value.

Rock pool fish use a combination of colour change and substrate choice to improve camouflage

Camouflage can be achieved by both morphological (e.g. colour, brightness and pattern change) and behavioural (e.g. substrate preference) means. Much of the research on behavioural background matching has been conducted on species with fixed coloration and body patterns, while less is known about the role background choice plays in species capable of rapid (within minutes or seconds) colour change. One candidate species is the rock goby, Gobius paganellus, a common rock pool fish capable of rapid changes in colour and brightness when placed on different backgrounds. However, their ability to match different backgrounds is not unbounded, with some colours and brightness being easier to match than others, thus raising the possibility that gobies may use behavioural background matching to make up for their limited ability to match certain backgrounds. We used digital image analysis and a model of predator vision to investigate the ability of rock gobies to match chromatic (beige and greenish-grey) and achromatic (varying brightness) backgrounds. We then conducted choice experiments to determine whether gobies exhibited a behavioural preference for the backgrounds they were best at matching. Gobies rapidly changed their colour and brightness when placed on the different backgrounds. However, the level of camouflage differed between backgrounds: fish were better at matching beige than greenish-grey, and darker than lighter backgrounds. When given the choice, gobies displayed a behavioural preference for the backgrounds they were best at matching. Our findings therefore show that rock gobies, and probably other animals, use a combination of morphological and behavioural means to achieve camouflage and in doing so mitigate limitations in either approach alone.