Justine J. Allen

Monitoring intracellular calcium is central to understanding cell signaling across nearly all cell types and organisms. Fluorescent genetically encoded calcium indicators (GECIs) remain the standard tools for in vivo calcium imaging, but require intense excitation light, leading to photobleaching, background autofluorescence and phototoxicity. Bioluminescent GECIs, which generate light enzymatically, eliminate these artifacts but have been constrained by low dynamic range and suboptimal calcium affinities. Here we show that CaBLAM (‘calcium bioluminescence activity monitor’), an engineered bioluminescent calcium indicator, achieves an order-of-magnitude improvement in signal contrast and a tunable affinity matched to physiological cytosolic calcium. CaBLAM enables single-cell and subcellular activity imaging at video frame rates in cultured neurons and sustained imaging over hours in awake, behaving animals. These capabilities establish CaBLAM as a robust and general alternative to fluorescent GECIs, extending calcium imaging to regimes where excitation light is undesirable or infeasible.

Measuring ongoing cellular activity is essential to understanding the dynamic functions of biological organisms. The most popular current approach is imaging fluorescence-based genetically encoded Ca ²⁺ indicators (GECIs). While fluorescent probes are useful in many contexts, bioluminescence-based GECIs—probes that generate light through oxidation of a small-molecule by a luciferase or photoprotein—have several distinct advantages. Because bioluminescent (BL) GECIs do not use the bright extrinsic excitation light required for fluorescence, BL GECIs do not photobleach, do not suffer from nonspecific autofluorescent background, and do not cause phototoxicity. Further, BL GECIs can be applied in contexts where directly shining photons on an imaging target is not possible. Despite these advantages, the use of BL GECIs has to date been limited by their small changes in bioluminescence intensity, high baseline signal at resting Ca ²⁺ concentrations, and suboptimal Ca ²⁺ affinities. Here, we describe a new BL GECI, CaBLAM ( Ca ²⁺ B io L uminescence A ctivity M onitor), that displays much higher dynamic range than previous BL GECIs and has a Ca ²⁺ affinity suitable for capturing physiological changes in cytosolic Ca ²⁺ concentration. With these improvements, CaBLAM captures single-cell and subcellular resolution activity at high frame rates in cultured neurons and in vivo , and allows multi-hour recordings in mice and behaving zebrafish. This new advance provides a robust alternative to traditional fluorescent GECIs that can enable or enhance imaging across many experimental conditions.

Male cuttlefish compete for females with a repertoire of visually dramatic behaviors. Laboratory experiments have explored this system in Sepia officinalis, but corroborative field data have eluded collection attempts by many researchers. While scuba diving in Turkey, we fortuitously filmed an intense sequence of consort/intruder behaviors in which the consort lost and then regained his female mate from the intruder. These agonistic bouts escalated in stages, leading to fast dramatic expression of the elaborate intense zebra display and culminating in biting and inking as the intruder male attempted a forced copulation of the female. When analyzed in the context of game theory, the patterns of fighting behavior were more consistent with mutual assessment than self-assessment of fighting ability. Additional observations of these behaviors in nature are needed to conclusively determine which models best represent conflict resolution, but our field observations agree with laboratory findings and provide a valuable perspective.

Unlike birds and insects, bats fly with wings composed of thin skin that envelops the bones of the forelimb and spans the area between the limbs, digits, and sometimes the tail. This skin is complex and unusual; it is thinner than typical mammalian skin and contains organized bundles of elastin and embedded skeletal muscles. These elements are likely responsible for controlling the shape of the wing during flight and contributing to the aerodynamic capabilities of bats. We examined the arrangement of two macroscopic architectural elements in bat wings, elastin bundles and wing membrane muscles, to assess the diversity in bat wing skin morphology. We characterized the plagiopatagium and dactylopatagium of 130 species from 17 families of bats using cross‐polarized light imaging. This method revealed structures with distinctive relative birefringence, heterogeneity of birefringence, variation in size, and degree of branching. We used previously published anatomical studies and tissue histology to identify birefringent structures, and we analyzed their architecture across taxa. Elastin bundles, muscles, neurovasculature, and collagenous fibers are present in all species. Elastin bundles are oriented in a predominantly spanwise or proximodistal direction, and there are five characteristic muscle arrays that occur within the plagiopatagium, far more muscle than typically recognized. These results inform recent functional studies of wing membrane architecture, support the functional hypothesis that elastin bundles aid wing folding and unfolding, and further suggest that all bats may use these architectural elements for flight. All species also possess numerous muscles within the wing membrane, but the architecture of muscle arrays within the plagiopatagium varies among families. To facilitate present and future discussion of these muscle arrays, we refine wing membrane muscle nomenclature in a manner that reflects this morphological diversity. The architecture of the constituents of the skin of the wing likely plays a key role in shaping wings during flight.

The slender filefish is a master of adaptive camouflage and can change its appearance within 1–3 s. Videos and photographs of this animal's cryptic bo

Commercial off-the-shelf digital cameras are inexpensive and easy-to-use instruments that can be used for quantitative scientific data acquisition if images are captured in raw format and processed so that they maintain a linear relationship with scene radiance. Here we describe the image-processing steps required for consistent data acquisition with color cameras. In addition, we present a method for scene-specific color calibration that increases the accuracy of color capture when a scene contains colors that are not well represented in the gamut of a standard color-calibration target. We demonstrate applications of the proposed methodology in the fields of biomedical engineering, artwork photography, perception science, marine biology, and underwater imaging.

A major component of cephalopod adaptive camouflage behavior has rarely been studied: their ability to change the three‐dimensionality of their skin by morphing their malleable dermal papillae. Recent work has established that simple, conical papillae in cuttlefish (Sepia officinalis) function as muscular hydrostats; that is, the muscles that extend a papilla also provide its structural support. We used brightfield and scanning electron microscopy to investigate and compare the functional morphology of nine types of papillae of different shapes, sizes and complexity in six species: S. officinalis small dorsal papillae, Octopus vulgaris small dorsal and ventral eye papillae, Macrotritopus defilippi dorsal eye papillae, Abdopus aculeatus major mantle papillae, O. bimaculoides arm, minor mantle, and dorsal eye papillae, and S. apama face ridge papillae. Most papillae have two sets of muscles responsible for extension: circular dermal erector muscles arranged in a concentric pattern to lift the papilla away from the body surface and horizontal dermal erector muscles to pull the papilla's perimeter toward its core and determine shape. A third set of muscles, retractors, appears to be responsible for pulling a papilla's apex down toward the body surface while stretching out its base. Connective tissue infiltrated with mucopolysaccharides assists with structural support. S. apama face ridge papillae are different: the contraction of erector muscles perpendicular to the ridge causes overlying tissues to buckle. In this case, mucopolysaccharide‐rich connective tissue provides structural support. These six species possess changeable papillae that are diverse in size and shape, yet with one exception they share somewhat similar functional morphologies. Future research on papilla morphology, biomechanics and neural control in the many unexamined species of octopus and cuttlefish may uncover new principles of actuation in soft, flexible tissue. J. Morphol. 275:371–390, 2014.

Coleoid cephalopods adaptively change their body patterns (color, contrast, locomotion, posture, and texture) for camouflage and signaling. Benthic octopuses and cuttlefish possess the capability, unique in the animal kingdom, to dramatically and quickly change their skin from smooth and flat to rugose and three‐dimensional. The organs responsible for this physical change are the skin papillae, whose biomechanics have not been investigated. In this study, small dorsal papillae from cuttlefish (Sepia officinalis) were preserved in their retracted or extended state, and examined with a variety of histological techniques including brightfield, confocal, and scanning electron microscopy. Analyses revealed that papillae are composed of an extensive network of dermal erector muscles, some of which are arranged in concentric rings while others extend across each papilla's diameter. Like cephalopod arms, tentacles, and suckers, skin papillae appear to function as muscular hydrostats. The collective action of dermal erector muscles provides both movement and structural support in the absence of rigid supporting elements. Specifically, concentric circular dermal erector muscles near the papilla's base contract and push the overlying tissue upward and away from the mantle surface, while horizontally arranged dermal erector muscles pull the papilla's perimeter toward its center and determine its shape. Each papilla has a white tip, which is produced by structural light reflectors (leucophores and iridophores) that lie between the papilla's muscular core and the skin layer that contains the pigmented chromatophores. In extended papillae, the connective tissue layer appeared thinner above the papilla's apex than in surrounding areas. This result suggests that papilla extension might create tension in the overlying connective tissue and chromatophore layers, storing energy for elastic retraction. Numerous, thin subepidermal muscles form a meshwork between the chromatophore layer and the epidermis and putatively provide active papillary retraction. J. Morphol., 2013.

Cephalopods are renowned for their ability to adaptively camouflage on diverse backgrounds. Sepia officinalis camouflage body patterns have been characterized spectrally in the laboratory but not in the field due to the challenges of dynamic natural light fields and the difficulty of using spectrophotometric instruments underwater. To assess cuttlefish color match in their natural habitats, we studied the spectral properties of S. officinalis and their backgrounds on the Aegean coast of Turkey using point-by-point in situ spectrometry. Fifteen spectrometry datasets were collected from seven cuttlefish; radiance spectra from animal body components and surrounding substrates were measured at depths shallower than 5 m. We quantified luminance and color contrast of cuttlefish components and background substrates in the eyes of hypothetical di- and trichromatic fish predators. Additionally, we converted radiance spectra to sRGB color space to simulate their in situ appearance to a human observer. Within the range of natural colors at our study site, cuttlefish closely matched the substrate spectra in a variety of body patterns. Theoretical calculations showed that this effect might be more pronounced at greater depths. We also showed that a non-biological method (“Spectral Angle Mapper”), commonly used for spectral shape similarity assessment in the field of remote sensing, shows moderate correlation to biological measures of color contrast. This performance is comparable to that of a traditional measure of spectral shape similarity, hue and chroma. This study is among the first to quantify color matching of camouflaged cuttlefish in the wild.