Elementary response triggered by transducin in retinal rods.

G protein-coupled receptor (GPCR) signaling is crucial for many physiological processes. A signature of such pathways is high amplification, a concept originating from retinal rod phototransduction, whereby one photoactivated rhodopsin molecule (Rho*) was long reported to activate several hundred transducins (GT*s), each then activating a cGMP-phosphodiesterase catalytic subunit (GT*·PDE*). This high gain at the Rho*-to-GT* step has been challenged more recently, but estimates remain dispersed and rely on some nonintact rod measurements. With two independent approaches, one with an extremely inefficient mutant rhodopsin and the other with WT bleached rhodopsin, which has exceedingly weak constitutive activity in darkness, we obtained an estimate for the electrical effect from a single GT*·PDE* molecular complex in intact mouse rods. Comparing the single-GT*·PDE* effect to the WT single-photon response, both in Gcaps-/- background, gives an effective gain of only ∼12-14 GT*·PDE*s produced per Rho*. Our findings have finally dispelled the entrenched concept of very high gain at the receptor-to-G protein/effector step in GPCR systems.

Voltage-sensitive conductances increase the sensitivity of rod photoresponses following pigment bleaching.

KEY POINTS: Following substantial bleaching of the visual pigment, the desensitization of the rod photovoltage is not as substantial as the desensitization of the rod outer segment photocurrent. The block of cation conductances during the internal dialysis of Cs+ further desensitizes the photovoltage thereby eliminating its difference in desensitization with the rod outer segment photocurrent. Bleached visual pigment produced an acceleration of the rod photovoltage with respect to the outer segment photocurrent, which is eliminated upon internal dialysis of Cs+ . ABSTRACT: A majority of our visual experience occurs during the day when a substantial fraction of the visual pigment in our photoreceptor cells is bleached. Under these conditions it is widely believed that rods are saturated and do not contribute substantially to downstream signalling. However, behavioural experiments on subjects with only rod function reveals that these individuals unexpectedly retain substantial vision in daylight. We sought to understand this discrepancy by characterizing the sensitivity of rod photoresponses following exposure to bright bleaching light. Measurements of the rod outer segment photocurrent in transgenic mice, which have only rod function, revealed the well-studied reduction in the sensitivity of rod photoresponses following pigment bleaching. However, membrane voltage measurements showed that the desensitization of the photovoltage was considerably less than that of the outer segment photocurrent following equivalent pigment bleaching. This discrepancy was largely eliminated during the blockade of cation channels due to the internal dialysis of Cs+ , which increased the bleach-induced desensitization of the photovoltage and slowed its temporal characteristics. Thus, sensitization of the photovoltage by rod inner segment conductances appears to extend the operating range of rod phototransduction following pigment bleaching.

The retina visual cycle is driven by cis retinol oxidation in the outer segments of cones.

Vertebrate rod and cone photoreceptors require continuous supply of chromophore for regenerating their visual pigments after photoactivation. Cones, which mediate our daytime vision, demand a particularly rapid supply of 11-cis retinal chromophore in order to maintain their function in bright light. An important contribution to this process is thought to be the chromophore precursor 11-cis retinol, which is supplied to cones from Müller cells in the retina and subsequently oxidized to 11-cis retinal as part of the retina visual cycle. However, the molecular identity of the cis retinol oxidase in cones remains unclear. Here, as a first step in characterizing this enzymatic reaction, we sought to determine the subcellular localization of this activity in salamander red cones. We found that the onset of dark adaptation of isolated salamander red cones was substantially faster when exposing directly their outer vs. their inner segment to 9-cis retinol, an analogue of 11-cis retinol. In contrast, this difference was not observed when treating the outer vs. inner segment with 9-cis retinal, a chromophore analogue which can directly support pigment regeneration. These results suggest, surprisingly, that the cis-retinol oxidation occurs in the outer segments of cone photoreceptors. Confirming this notion, pigment regeneration with exogenously added 9-cis retinol was directly observed in the truncated outer segments of cones, but not in rods. We conclude that the enzymatic machinery required for the oxidation of recycled cis retinol as part of the retina visual cycle is present in the outer segments of cones.

Chromophore supply rate-limits mammalian photoreceptor dark adaptation.

Efficient regeneration of visual pigment following its destruction by light is critical for the function of mammalian photoreceptors. Here, we show that misexpression of a subset of cone genes in the rd7 mouse hybrid rods enables them to access the normally cone-specific retina visual cycle. The rapid supply of chromophore by the retina visual cycle dramatically accelerated the mouse rod dark adaptation. At the same time, the competition between rods and cones for retina-derived chromophore slowed cone dark adaptation, indicating that the cone specificity of the retina visual cycle is key for rapid cone dark adaptation. Our findings demonstrate that mammalian photoreceptor dark adaptation is dominated by the supply of chromophore. Misexpression of cone genes in rods may represent a novel approach to treating visual disorders associated with mutations of visual cycle proteins or with reduced retinal pigment epithelium function due to aging.

Bicarbonate boosts flash response amplitude to augment absolute sensitivity and extend dynamic range in murine retinal rods.

Rod photoreceptors in the retina adjust their responsiveness and sensitivity so that they can continue to provide meaningful information over a wide range of light intensities. By stimulating membrane guanylate cyclases in the outer segment to synthesize cGMP at a faster rate in a Ca2+-dependent fashion, bicarbonate increases the circulating "dark" current and accelerates flash response kinetics in amphibian rods. Compared to amphibian rods, mammalian rods are smaller in size, operate at a higher temperature, and express visual cascade proteins with somewhat different biochemical properties. Here, we evaluated the role of bicarbonate in rods of cpfl3 mice. These mice are deficient in their expression of functional cone transducin, Gnat2, making cones very insensitive to light, so the rod response to light could be observed in isolation in electroretinogram recordings. Bicarbonate increased the dark current and absolute sensitivity and quickened flash response recovery in mouse rods to a greater extent than in amphibian rods. In addition, bicarbonate enabled mouse rods to respond over a range that extended to dimmer flashes. Larger flash responses may have resulted in part from a bicarbonate-induced elevation in intracellular pH. However, high pH alone had little effect on flash response recovery kinetics and even suppressed the accelerating effect of bicarbonate, consistent with a direct, modulatory action of bicarbonate on Ca2+- dependent, membrane guanylate cyclase activity.

Apo-Opsin and Its Dark Constitutive Activity across Retinal Cone Subtypes.

Retinal rod and cone photoreceptors mediate vision in dim and bright light, respectively, by transducing absorbed photons into neural electrical signals. Their phototransduction mechanisms are essentially identical. However, one difference is that, whereas a rod visual pigment remains stable in darkness, a cone pigment has some tendency to dissociate spontaneously into apo-opsin and retinal (the chromophore) without isomerization. This cone-pigment property is long known but has mostly been overlooked. Importantly, because apo-opsin has weak constitutive activity, it triggers transduction to produce electrical noise even in darkness. Currently, the precise dark apo-opsin contents across cone subtypes are mostly unknown, as are their dark activities. We report here a study of goldfish red (L), green (M), and blue (S) cones, finding with microspectrophotometry widely different apo-opsin percentages in darkness, being ∼30% in L cones, ∼3% in M cones, and negligible in S cones. L and M cones also had higher dark apo-opsin noise than holo-pigment thermal isomerization activity. As such, given the most likely low signal amplification at the pigment-to-transducin/phosphodiesterase phototransduction step, especially in L cones, apo-opsin noise may not be easily distinguishable from light responses and thus may affect cone vision near threshold.

Breaking the covalent bond--a pigment property that contributes to desensitization in cones.

Retinal rod and cone pigments consist of an apoprotein, opsin, covalently linked to a chromophore, 11-cis retinal. Here we demonstrate that the formation of the covalent bond between opsin and 11-cis retinal is reversible in darkness in amphibian red cones, but essentially irreversible in red rods. This dissociation, apparently a general property of cone pigments, results in a surprisingly large amount of free opsin--about 10% of total opsin--in dark-adapted red cones. We attribute this significant level of free opsin to the low concentration of intracellular free 11-cis retinal, estimated to be only a tiny fraction (approximately 0.1 %) of the pigment content in red cones. With its constitutive transducin-stimulating activity, the free cone opsin produces an approximately 2-fold desensitization in red cones, equivalent to that produced by a steady light causing 500 photoisomerizations s-1. Cone pigment dissociation therefore contributes to the sensitivity difference between rods and cones.

Night blindness and the mechanism of constitutive signaling of mutant G90D rhodopsin.

The G90D rhodopsin mutation is known to produce congenital night blindness in humans. This mutation produces a similar condition in mice, because rods of animals heterozygous (D+) or homozygous (D+/+) for this mutation have decreased dark current and sensitivity, reduced Ca(2+), and accelerated values of tau(REC) and tau(D), similar to light-adapted wild-type (WT) rods. Our experiments indicate that G90D pigment activates the cascade, producing an equivalent background light of approximately 130 Rh* rod(-1) for D+ and 890 Rh* rod(-1) for D+/+. The active species of the G90D pigment could be unregenerated G90D opsin or G90D rhodopsin, either spontaneously activated (as Rh*) or in some other form. Addition of 11-cis-retinal in lipid vesicles, which produces regeneration of both WT and G90D opsin in intact rods and ROS membranes, had no effect on the waveform or sensitivity of dark-adapted G90D responses, indicating that the active species is not G90D opsin. The noise spectra of dark-adapted G90D and WT rods are similar, and the G90D noise variance is much less than of a WT rod exposed to background light of about the same intensity as the G90D equivalent light, indicating that Rh* is not the active species. We hypothesize that G90D rhodopsin undergoes spontaneous changes in molecular conformation which activate the transduction cascade with low gain. Our experiments provide the first indication that a mutant form of the rhodopsin molecule bound to its 11-cis-chromophore can stimulate the visual cascade spontaneously at a rate large enough to produce visual dysfunction.

Isoelectric Focusing to Quantify Rhodopsin Phosphorylation in Mouse Retina.

Rhodopsin is a G-protein coupled receptor (GPCR) that mediates vision under dim light. Upon light exposure, rhodopsin is phosphorylated at multiple serine and threonine sites at its carboxyl-terminus by rhodopsin kinase (GRK1). This, in turn, reduces its ability to activate the visual G-protein transducin. Binding of light-activated, phosphorylated rhodopsin by arrestin (ARR1) fully terminates the catalytic activity of rhodopsin. Quantification of the levels of the differentially phosphorylated rhodopsin species provides definitive information about the role of phosphorylated rhodopsin in visual functions. Isoelectric Focusing (IEF) is a technique which is used to separate ampholytic components, such as proteins, based on their isoelectric point (pI). It is a useful technique used to distinguish protein isoforms and post-translational modifications such as phosphorylation, glycosylation, deamination, and acetylation, due to their effects on the protein's pI. Isoelectric Focusing can provide high resolution of differentially phosphorylated forms of a protein. Though other techniques such as kinase activity assays, phospho-specific antibodies, western blot, enzyme-linked immunosorbent assays (ELISA), radiolabeling and mass spectrometry are used to detect and quantify protein phosphorylation, IEF is a simple and cost-effective method to quantify rhodopsin phosphorylation, as it can readily detect individual phosphorylated forms. Here we provide a detailed protocol for determining phosphorylated rhodopsin species using the Isoelectric Focusing technique.

Opsin activation as a cause of congenital night blindness.

Three different mutations of rhodopsin are known to cause autosomal dominant congenital night blindness in humans. Although the mutations have been studied for 10 years, the molecular mechanism of the disease is still a subject of controversy. We show here, using a transgenic Xenopus laevis model, that the photoreceptor cell desensitization that is a hallmark of the disease results from persistent signaling by constitutively active mutant opsins.

Turning cones off: the role of the 9-methyl group of retinal in red cones.

Our ability to see in bright light depends critically on the rapid rate at which cone photoreceptors detect and adapt to changes in illumination. This is achieved, in part, by their rapid response termination. In this study, we investigate the hypothesis that this rapid termination of the response in red cones is dependent on interactions between the 9-methyl group of retinal and red cone opsin, which are required for timely metarhodopsin (Meta) II decay. We used single-cell electrical recordings of flash responses to assess the kinetics of response termination and to calculate guanylyl cyclase (GC) rates in salamander red cones containing native visual pigment as well as visual pigment regenerated with 11-cis 9-demethyl retinal, an analogue of retinal in which the 9-methyl group is missing. After exposure to bright light that photoactivated more than approximately 0.2% of the pigment, red cones containing the analogue pigment had a slower recovery of both flash response amplitudes and GC rates (up to 10 times slower at high bleaches) than red cones containing 11-cis retinal. This finding is consistent with previously published biochemical data demonstrating that red cone opsin regenerated in vitro with 11-cis 9-demethyl retinal exhibited prolonged activation as a result of slowed Meta II decay. Our results suggest that two different mechanisms regulate the recovery of responsiveness in red cones after exposure to light. We propose a model in which the response recovery in red cones can be regulated (particularly at high light intensities) by the Meta II decay rate if that rate has been inhibited. In red cones, the interaction of the 9-methyl group of retinal with opsin promotes efficient Meta II decay and, thus, the rapid rate of recovery.

Visual cycle: Dependence of retinol production and removal on photoproduct decay and cell morphology.

The visual cycle is a chain of biochemical reactions that regenerate visual pigment following exposure to light. Initial steps, the liberation of all-trans retinal and its reduction to all-trans retinol by retinol dehydrogenase (RDH), take place in photoreceptors. We performed comparative microspectrophotometric and microfluorometric measurements on a variety of rod and cone photoreceptors isolated from salamander retinae to correlate the rates of photoproduct decay and retinol production. Metapigment decay rate was spatially uniform within outer segments and 50-70 times faster in the cells that contained cone-type pigment (SWS2 and M/LWS) compared to cells with rod-type pigment (RH1). Retinol production rate was strongly position dependent, fastest at the base of outer segments. Retinol production rate was 10-40 times faster in cones with cone pigments (SWS2 and M/LWS) than in the basal OS of rods containing rod pigment (RH1). Production rate was approximately five times faster in rods containing cone pigment (SWS2) than the rate in basal OS of rods containing the rod pigment (RH1). We show that retinol production is defined either by metapigment decay rate or RDH reaction rate, depending on cell type or outer segment region, whereas retinol removal is defined by the surface-to-volume ratio of the outer segment and the availability of retinoid binding protein (IRBP). The more rapid rates of retinol production in cones compared to rods are consistent with the more rapid operation of the visual cycle in these cells.

Cyclic nucleotide-gated ion channels in rod photoreceptors are protected from retinoid inhibition.

In vertebrate rods, photoisomerization of the 11-cis retinal chromophore of rhodopsin to the all-trans conformation initiates a biochemical cascade that closes cGMP-gated channels and hyperpolarizes the cell. All-trans retinal is reduced to retinol and then removed to the pigment epithelium. The pigment epithelium supplies fresh 11-cis retinal to regenerate rhodopsin. The recent discovery that tens of nanomolar retinal inhibits cloned cGMP-gated channels at low [cGMP] raised the question of whether retinoid traffic across the plasma membrane of the rod might participate in the signaling of light. Native channels in excised patches from rods were very sensitive to retinoid inhibition. Perfusion of intact rods with exogenous 9- or 11-cis retinal closed cGMP-gated channels but required higher than expected concentrations. Channels reopened after perfusing the rod with cellular retinoid binding protein II. PDE activity, flash response kinetics, and relative sensitivity were unchanged, ruling out pharmacological activation of the phototransduction cascade. Bleaching of rhodopsin to create all-trans retinal and retinol inside the rod did not produce any measurable channel inhibition. Exposure of a bleached rod to 9- or 11-cis retinal did not elicit channel inhibition during the period of rhodopsin regeneration. Microspectrophotometric measurements showed that exogenous 9- or 11-cis retinal rapidly cross the plasma membrane of bleached rods and regenerate their rhodopsin. Although dark-adapted rods could also take up large quantities of 9-cis retinal, which they converted to retinol, the time course was slow. Apparently cGMP-gated channels in intact rods are protected from the inhibitory effects of retinoids that cross the plasma membrane by a large-capacity buffer. Opsin, with its chromophore binding pocket occupied (rhodopsin) or vacant, may be an important component. Exceptionally high retinoid levels, e.g., associated with some retinal degenerations, could overcome the buffer, however, and impair sensitivity or delay the recovery after exposure to bright light.

Spontaneous activation of visual pigments in relation to openness/closedness of chromophore-binding pocket.

Visual pigments can be spontaneously activated by internal thermal energy, generating noise that interferes with real-light detection. Recently, we developed a physicochemical theory that successfully predicts the rate of spontaneous activity of representative rod and cone pigments from their peak-absorption wavelength (λmax), with pigments having longer λmax being noisier. Interestingly, cone pigments may generally be ~25 fold noisier than rod pigments of the same λmax, possibly ascribed to an 'open' chromophore-binding pocket in cone pigments defined by the capability of chromophore-exchange in darkness. Here, we show in mice that the λmax-dependence of pigment noise could be extended even to a mutant pigment, E122Q-rhodopsin. Moreover, although E122Q-rhodopsin shows some cone-pigment-like characteristics, its noise remained quantitatively predictable by the 'non-open' nature of its chromophore-binding pocket as in wild-type rhodopsin. The openness/closedness of the chromophore-binding pocket is potentially a useful indicator of whether a pigment is intended for detecting dim or bright light.

Cambrian origin of the CYP27C1-mediated vitamin A1-to-A2 switch, a key mechanism of vertebrate sensory plasticity.

The spectral composition of ambient light varies across both space and time. Many species of jawed vertebrates adapt to this variation by tuning the sensitivity of their photoreceptors via the expression of CYP27C1, an enzyme that converts vitamin A1 into vitamin A2, thereby shifting the ratio of vitamin A1-based rhodopsin to red-shifted vitamin A2-based porphyropsin in the eye. Here, we show that the sea lamprey (Petromyzon marinus), a jawless vertebrate that diverged from jawed vertebrates during the Cambrian period (approx. 500 Ma), dynamically shifts its photoreceptor spectral sensitivity via vitamin A1-to-A2 chromophore exchange as it transitions between photically divergent aquatic habitats. We further show that this shift correlates with high-level expression of the lamprey orthologue of CYP27C1, specifically in the retinal pigment epithelium as in jawed vertebrates. Our results suggest that the CYP27C1-mediated vitamin A1-to-A2 switch is an evolutionarily ancient mechanism of sensory plasticity that appeared not long after the origin of vertebrates.

Rhodopsin kinase and arrestin binding control the decay of photoactivated rhodopsin and dark adaptation of mouse rods.

Photoactivation of vertebrate rhodopsin converts it to the physiologically active Meta II (R*) state, which triggers the rod light response. Meta II is rapidly inactivated by the phosphorylation of C-terminal serine and threonine residues by G-protein receptor kinase (Grk1) and subsequent binding of arrestin 1 (Arr1). Meta II exists in equilibrium with the more stable inactive form of rhodopsin, Meta III. Dark adaptation of rods requires the complete thermal decay of Meta II/Meta III into opsin and all-trans retinal and the subsequent regeneration of rhodopsin with 11-cis retinal chromophore. In this study, we examine the regulation of Meta III decay by Grk1 and Arr1 in intact mouse rods and their effect on rod dark adaptation. We measure the rates of Meta III decay in isolated retinas of wild-type (WT), Grk1-deficient (Grk1(-/-)), Arr1-deficient (Arr1(-/-)), and Arr1-overexpressing (Arr1(ox)) mice. We find that in WT mouse rods, Meta III peaks ∼6 min after rhodopsin activation and decays with a time constant (τ) of 17 min. Meta III decay slows in Arr1(-/-) rods (τ of ∼27 min), whereas it accelerates in Arr1(ox) rods (τ of ∼8 min) and Grk1(-/-) rods (τ of ∼13 min). In all cases, regeneration of rhodopsin with exogenous 11-cis retinal is rate limited by the decay of Meta III. Notably, the kinetics of rod dark adaptation in vivo is also modulated by the levels of Arr1 and Grk1. We conclude that, in addition to their well-established roles in Meta II inactivation, Grk1 and Arr1 can modulate the kinetics of Meta III decay and rod dark adaptation in vivo.

Complementary shifts in photoreceptor spectral tuning unlock the full adaptive potential of ultraviolet vision in birds.

Color vision in birds is mediated by four types of cone photoreceptors whose maximal sensitivities (λmax) are evenly spaced across the light spectrum. In the course of avian evolution, the λmax of the most shortwave-sensitive cone, SWS1, has switched between violet (λmax > 400 nm) and ultraviolet (λmax < 380 nm) multiple times. This shift of the SWS1 opsin is accompanied by a corresponding short-wavelength shift in the spectrally adjacent SWS2 cone. Here, we show that SWS2 cone spectral tuning is mediated by modulating the ratio of two apocarotenoids, galloxanthin and 11',12'-dihydrogalloxanthin, which act as intracellular spectral filters in this cell type. We propose an enzymatic pathway that mediates the differential production of these apocarotenoids in the avian retina, and we use color vision modeling to demonstrate how correlated evolution of spectral tuning is necessary to achieve even sampling of the light spectrum and thereby maintain near-optimal color discrimination.

Physiological and microfluorometric studies of reduction and clearance of retinal in bleached rod photoreceptors.

The visual cycle comprises a sequence of reactions that regenerate the visual pigment in photoreceptors during dark adaptation, starting with the reduction of all-trans retinal to all-trans retinol and its clearance from photoreceptors. We have followed the reduction of retinal and clearance of retinol within bleached outer segments of red rods isolated from salamander retina by measuring its intrinsic fluorescence. Following exposure to a bright light (bleach), increasing fluorescence intensity was observed to propagate along the outer segments in a direction from the proximal region adjacent to the inner segment toward the distal tip. Peak retinol fluorescence was achieved after approximately 30 min, after which it declined very slowly. Clearance of retinol fluorescence is considerably accelerated by the presence of the exogenous lipophilic substances IRBP (interphotoreceptor retinoid binding protein) and serum albumin. We have used simultaneous fluorometric and electrophysiological measurements to compare the rate of reduction of all-trans retinal to all-trans retinol to the rate of recovery of flash response amplitude in these cells in the presence and absence of IRBP. We find that flash response recovery in rods is modestly accelerated in the presence of extracellular IRBP. These results suggest such substances may participate in the clearance of retinoids from rod photoreceptors, and that this clearance, at least in rods, may facilitate dark adaptation by accelerating the clearance of photoproducts of bleaching.

Optics of cone photoreceptors in the chicken (Gallus gallus domesticus).

Vision is the primary sensory modality of birds, and its importance is evident in the sophistication of their visual systems. Coloured oil droplets in the cone photoreceptors represent an adaptation in the avian retina, acting as long-pass colour filters. However, we currently lack understanding of how the optical properties and morphology of component structures (e.g. oil droplet, mitochondrial ellipsoid and outer segment) of the cone photoreceptor influence the transmission of light into the outer segment and the ultimate effect they have on receptor sensitivity. In this study, we use data from microspectrophotometry, digital holographic microscopy and electron microscopy to inform electromagnetic models of avian cone photoreceptors to quantitatively investigate the integrated optical function of the cell. We find that pigmented oil droplets primarily function as spectral filters, not light collection devices, although the mitochondrial ellipsoid improves optical coupling between the inner segment and oil droplet. In contrast, unpigmented droplets found in violet-sensitive cones double sensitivity at its peak relative to other cone types. Oil droplets and ellipsoids both narrow the angular sensitivity of single cone photoreceptors, but not as strongly as those in human cones.

A complex carotenoid palette tunes avian colour vision.

The brilliantly coloured cone oil droplets of the avian retina function as long-pass cut-off filters that tune the spectral sensitivity of the photoreceptors and are hypothesized to enhance colour discrimination and improve colour constancy. Although it has long been known that these droplets are pigmented with carotenoids, their precise composition has remained uncertain owing to the technical challenges of measuring these very small, dense and highly refractile optical organelles. In this study, we integrated results from high-performance liquid chromatography, hyperspectral microscopy and microspectrophotometry to obtain a comprehensive understanding of oil droplet carotenoid pigmentation in the chicken (Gallus gallus). We find that each of the four carotenoid-containing droplet types consists of a complex mixture of carotenoids, with a single predominant carotenoid determining the wavelength of the spectral filtering cut-off. Consistent with previous reports, we find that the predominant carotenoid type in the oil droplets of long-wavelength-sensitive, medium-wavelength-sensitive and short-wavelength-sensitive type 2 cones are astaxanthin, zeaxanthin and galloxanthin, respectively. In addition, the oil droplet of the principal member of the double cone contains a mixture of galloxanthin and two hydroxycarotenoids (lutein and zeaxanthin). Short-wavelength-absorbing apocarotenoids are present in all of the droplet types, providing filtering of light in a region of the spectrum where filtering by hydroxy- and ketocarotenoids may be incomplete. Thus, birds rely on a complex palette of carotenoid pigments within their cone oil droplets to achieve finely tuned spectral filtering.