Our recent Methods in Ecology and Evolution paper – ‘Imaging biological surface topography in situ and in vivo‘ – shows how to use gel-based profilometry to image various biological surfaces. To start you need to press a gel into a surface of interest. The bottom surface of the gel is coated in a paint to create an impression of the surface that has standard optical properties (not clear, shiny, or coloured). Then lights are shone on the gel at different angles and photographs are taken at six different lighting angles. These photographs allow us to study the surface in incredible detail. The following images give more information on how we can do this and the benefits of it.
Our first picture shows the peduncle and tail of a yellow perch (Perca flavescens) being pressed into a gel. We use a gel-based profilometry system manufactured by GelSight Inc. (http://www.gelsight.com/). Image: Dylan Wainwright.
The six greyscale photographs in this image are of the scales from the Hawaiian dascyllus (Dascyllus albisella). Each image has a different lighting angle and all six will be used to reconstruct the surface topography on this patch of scales. Imaging a surface is as fast as positioning the specimen and taking six photographs. No specimen preparation is required – this method can be done on clear, shiny, wet, and slimy surfaces! Images: Dylan Wainwright and the Freshwater and Marine Image Bank.
In this picture you can see the surface topography of Dascyllus albisella, reconstructed from the six greyscale images in the previous image. This image captures the lateral line, visible at the top of the image as a row of scales connected by a canal. Heights on this surface are shown as colours: the warmer the colours (oranges and reds), the higher the heights. The height range of this surface is just over 200 microns – the highest parts of the surface are over 200 microns higher than the lowest . Images: Dylan Wainwright and the Freshwater and Marine Image Bank.
Each reconstructed surface is made up of over 18 million three-dimensional points (x, y, and z). This allows for a substantial amount of digital zoom with the ability to still recover surface features. Above is an enlarged view of the posterior margin of a scale from Dascyllus albisella from the same image as the previous two slides. The posterior margin of this scale is made of ctenii, which are small interlocking spines that are present on the scales of many species of fish. Those at the margin are the longest and newest, with older ctenii becoming shortened and serving as a scaffold to interlock with newer ones. Images: Dylan Wainwright and the Freshwater and Marine Image Bank.
The three-dimensional topography data recovered by gel-based profilometry can help you make unique observations on the surface texture of biological surfaces, such as the armor-like ganoid scales of Polypterus endlicheri (see ‘Materials design principles of ancient fish armour’ by Bruet et al. http://go.nature.com/2ivXi8I for more information on poylpterus armor). Using software for surface analysis, height profile lines can be generated (shown above), along with a variety of roughness and surface measurements (not shown). This topographic data is crucial for understanding how biological surfaces interact with their environments. Images: Dylan Wainwright and George Albert Boulenger.
With gel-based profilometry, you can tune the gel properties to match even very soft surfaces, such as the epidermis and mucus that covers the scales of live fish. Above, we show a bluegill (Lepomis macrochirus) that was imaged with and without mucus. Without mucus, many surface details of scales are obvious, such as the concentric growth lines of each scale, the lateral line, and clear margins made of spiny ctenii. When mucus is present, the surface details are obscured. Below each image we provide tables of common surface parameters including root-mean-square roughness (Sq – http://bit.ly/2Amhpeb), kurtosis (Sku – http://bit.ly/2zUY8ne), and skew (Ssk – http://bit.ly/2zUY8ne). Roughness is much lower on the surface with mucus, demonstrating its smoothing effect. This smoothing effect and the material properties of mucus will likely affect the swimming performance of this fish, and these results show how useful this technique can be for exploring surfaces of live animals. Images: Dylan Wainwright and the Freshwater and Marine Image Bank.
Gel-based profilometry is non-invasive and only needs pressure to be applied to the surface of interest to get the image. Above is the surface topography of the back of a human hand. The pores are evident as small blue regions with low elevation. Long flexible structures like hairs will be pressed flat by the sampling gel, as seen in the hairs above. Image: Dylan Wainwright.
You can see the surface of a Boston fern (Nephrolepis exaltata) above. This image was taken at high magnification and then cropped to a 1 mm by 1 mm square. Stomata with guard cells are visible on the surface of the leaf as ring-shaped cells. Images: Dylan Wainwright and Marija Gajić (http://bit.ly/2AxryHp).
This is the forewing of a dragonfly. The wing venation pattern is obvious using this technique, and small spines are present on many of the veins, especially the distal veins towards the wing tip. We produced this image without any special preparation of the subject and without damaging these delicate wings. Images: Dylan Wainwright and Wellcome Library, London (http://bit.ly/2AkcT1J).
The above image shows a dorsal patch of skin from the Chinese crocodile lizard (Shinisaurus crocodilurus). This lizard is an endangered semiaquatic species with skin similar in appearance to a crocodiles (as its name suggests). Gel-based profilometry provides a non-destructive way of investigating the skin morphology of this species using museum specimens. Images: Dylan Wainwright and spacebirdy (CC-BY-SA-3.0) (http://bit.ly/2jCtvb4).
Above we have both a greyscale image and a height map from the hand of a Sulawesi lined gliding lizard (Draco splinotus). For two or one-dimensional measurements, greyscale images can be valuable because of their high contrast. Gel-based profilometry produces grayscale images at a range of sizes, comparable to low to medium magnification scanning electron microscopy. Images: Dylan Wainwright and A.S.Kono (http://bit.ly/2BCFY6W).
The denticles from the lateral flank of a leopard shark (Triakis semifasciata) were imaged and you can see the topographic reconstruction above. Denticles have been shown to increase swimming performance and understanding their surface topography is crucial for connecting the form of shark denticles to hydrodynamic function (see ‘The hydrodynamic function of shark skin and two biomimetic applications’ by Oeffner and Lauder, for example). Images: Dylan Wainwright and Tom Hilton (http://bit.ly/2BpW3vv).
This image shows the skin texture of the white marlin. Although most fish only have one type of bony structure in their skin (scales), white marlin have two. The first are larger, teardrop shaped scales with forked ends that are embedded in the dermis – they’re visible as larger impressions above. The second bony structure present on white marline skin are smaller peaks that are attached to the skin surface and look like small grains in the images above. Understanding these structures is an important step to understanding the function of marlin skin and the reasons behind these modifications (for more information on these scales see ‘Comparative morphology of the scales of roundscale spearfish Tetrapturus georgii and white marlin Kajikia albida’ by Loose et al. – http://bit.ly/2Bq5UBM). Images: Dylan Wainwright and public domain image.