Rob Campbell – Softbites

2021-01-202021-01-20

Squid reveal the secret to a “perfect” lens

Original paper: Eye patches: Protein assembly of index-gradient squid lenses

Evolution usually solves challenges differently than human engineers—something easy for biology is often difficult for us, and vice versa. Learning from biology can help us solve difficult challenges more easily. One example of this is making complex optical lenses.

**Figure 1.** Lenses use refraction and geometry to direct light. (Figure by the author)

When light enters a new material it refracts, changing velocity and direction. A lens uses geometry and material properties to direct light on a specific path. You can see in Figure 1 how the shape and refractive index of a biconvex lens combine to direct light at a single spot. In biology, complex eyes like those found in most vertebrates and in squid have a lens that directs light onto the retina at the back of the eye, forming an image to be processed by the brain. Squid use spherical lenses to do this, but spherical lenses have a problem. As you can see in Figure 2, if you make a spherical lens out of one material (like glass), the light rays overlap after exiting the lens and the resulting image is blurry. This is called “spherical aberration.” Human engineers use spherical lenses a lot, and we correct for spherical aberration by combining multiple lenses. Squid, on the other hand, have evolved a lens that self-corrects for this distortion.

**Figure 2.** Spherical lenses usually produce blurry images, but not in squid. (Figure by the author)

We know, in theory, how a squid might do this. In 1854, the famous physicist James Clerk Maxwell mathematically designed a spherical lens with “perfect” focus. He showed that if the density of the lens changes along the radius, forming a density gradient that he called a “perfect medium,” then the lens will produce a clear image. Today engineers can make gradient index lenses like this, but the process is difficult and energy intensive. Squid evolved to grow them easily. Could understanding how squid make these lenses help human engineers learn to do the same thing? This question inspired Dr. Jing Cai and Prof. Alison Sweeney to study the structure of the squid lens.

**Figure 3.** A squid lens has rings, which could combine to create a “perfect medium.” (Figure by the author, photograph from the *Museum of Museum of New Zealand Te Papa Tongarewa, as reported by* *Nerdist*)

You can see in Figure 3 that when you crack open the lens in a squid eye you find rings like the inside of a tree trunk. If each of these rings has a slightly different density, they could combine to create a perfect medium. The lens of an eye is made out of proteins called crystallins, which fold into individual particles before linking together into a single material. Cai and her collaborators discovered that the lenses of the Longfin inshore squid (Doryteuthis pealeii) use 53 different crystallin proteins of different sizes. They also found that the different proteins are used in different parts of the lens, and each layer of the lens has a slightly different structure. As you can see in Figure 4, small proteins at the center of the lens are densely packed together so that each protein is connected to six other proteins. However, the larger proteins at the edge of the lens have more space between them, and each protein only touches two others.

**Figure 4.** Protein particles assemble differently in different parts of the lens, creating a “perfect medium.” (Figure by the author)

This makes sense when you think about the cells that make these proteins. Cells rely on diffusion to bring building blocks to the right place for protein assembly and to send each assembled protein out to where it’s needed. When finished proteins link together to grow the lens, they disrupt this diffusion and stop protein production. By growing from dense to less dense and using so many different proteins (53 in the Longfin inshore squid), the cells are able to start and stop the growth of different layers while maintaining a single particle network. No part of the lens separates out or turns opaque, but there are still large enough regions with different densities to diffract light into alignment.

Cai and her collaborators showed that squid lenses definitely use a density gradient similar to Maxwell’s perfect medium to correct for spherical aberration. It’s likely that this density gradient not only creates a perfect medium, but also helps control lens assembly. Now that we know how squid build a perfect spherical lens, it is easier to envision how human engineers could grow our own complex optical materials.

2019-04-102019-11-26

Biological Materials at SICB 2019

It’s unusual to run a symposium as a PhD student, but anyone can do it! I was lucky to find a great mentor to guide me through the process. Together we organized 11 speakers, 2 workshops, and 11 poster presentations for a full day discussion on what soft matter, materials, and evolutionary biology have in common. From fire ants to spider silk, tooth enamel to lizard scales, and chemistry to computer science, there are lots of opportunities for soft-matter researchers to study biological questions.

The annual meeting of the Society for Integrative and Comparative Biology (SICB) is one of the core conferences for organismal biology. Originally called the “American Society of Zoologists,” the society changed its name to SICB in 1996 to emphasize the “integration” of different biological specializations. This commitment to interdisciplinary research made SICB the perfect home for my interest in biologically produced materials.

I’m interested in how biomaterials are created and diversify, a topic that draws on soft matter physics, mechanics, and evolutionary biology. There are a lot of exciting questions in this area, but because they are so interdisciplinary, there are not that many people who work on them. Interdisciplinary research often falls outside traditional departments and grant funding options, making these projects hard to design and run. They also require careful communication skills (if you talk to an engineer and an evolutionary biologist about the “evolution of a biomaterial” you might get two very different answers– the engineer might think of “material evolution” as a change during the material’s use (how does it respond to heat or light?), while the biologist might think about changes as the material developed with different organisms over millions of years). Nevertheless, I think interdisciplinary research questions are some of the most exciting and important, and luckily I’m not alone.

Together with my co-organizer, Dr. Mason Dean from the Biomaterials Department of the Max Planck Institute for Colloids and Interfaces, we organized the SICB symposium “Adaptation and Evolution of Biological Materials” (#AEBM #SICB2019) to highlight what is already being done in this field, and to encourage more biologists to start working with materials and soft matter.

Here are some highlights from our speakers:

Entanglement

Beyond “active matter” systems like fish schools or bird flocks, there are also collections of individual organisms that entangle together and behave like squishy, living materials. Prof. David Hu and Prof. Saad Bhamla presented on two different entangled soft matter systems: fire ant swarms and worm blobs. Both can act sometimes like a liquid and sometimes like a solid, depending on how the individuals link together. These systems can be described similarly to collections of molecules, complete with phase separation behavior!

These worm blobs are “active” and behave as viscoelastic fluids (both solid+liquid properties) — teaser video below. Come to the talk to learn more about these extreme biological system + active soft matter. https://t.co/iDYhPLkEri pic.twitter.com/MwlMcTdrmD
— Saad Bhamla (@BhamlaLab) January 2, 2019

Tunability

Unlike a lot of human-engineered systems, almost all biological materials have multiple functions. Dr. Beth Mortimer studies vibrational communication in spiders, worms, and elephants. Here she presented recent work suggesting the material vibration sensors built into spider legs might be tuned specifically for silk material properties — highlighting how silk has evolved to be both a structural and sensory material.

Assembly

Biological materials are famous for being made of simple, individual components that can assemble into complex structures on their own (i.e. “self assembly” without a human engineer). We had a lot of talks referencing this topic. Dr. Linnea Hesse studies the joints of branching plants to try and learn why they are so sturdy. She found that the vascular bundles that transport water (the equivalent of human capillaries for blood flow) adapt to external forces as the branch grows. This way the bottom of the branch is arranged differently than the top to optimize load bearing.

plant-branch-loading — The organization of vascular bundles in the dragon tree changes during growth, making the joints between branches and the trunk stronger. (Image courtesy of Dr. Linnea Hesse)

On a smaller scale, Prof. Matt Harrington presented on a new model of fiber formation from the velvet worm. Velvet worms shoot slime at their prey, which quickly hardens into fibers with strength comparable to nylon. If that wasn’t cool enough, these fibers can be dissolved in water and then later resolidify! Making them an intriguing model for new biodegradable plastics. Unlike spider silk, which is made of tiny highly ordered fibers, the “silk” of the velvet work seems to be made of relatively disordered charge-stabilized droplets.

Last but not least, Dr. Ainsley Seago has surveyed the colorful nanostructured scales of hundreds of species in two lineages of beetles. Her results suggest that even though these surfaces exhibit many different optical properties, they’re all likely assembled as liquids in a process remarkably similar to the assembly of cell membranes (called lyotropic assembly).

beetle-structural-color — The shiny, bright colors on beetles come from nanostructured scales. (Image courtesy of Dr. Ainsley Seago)

Image Analysis

Dr. Daniel Baum is an expert on computational solutions for automated image analysis. He presented on common approaches for automatically selecting different parts of an image. This is really useful for studying material and biological systems with lots of similar repeating structures, and modeling how these systems respond to external forces. He presented examples of this work applied to the study of sharks and rays, whose soft cartilaginous skeletons are wrapped in a network of tiny, repeating, mineralized plates (called tesserae).

modeling-tesserae — Computational methods, such as the watershed algorithm, can automatically segment different parts of an image and be used to construct 3D models of bones, cartilage, and other material. (Image courtesy of Dr. Daniel Baum)

Hierarchy

The layered organization of materials at different scales (forming a hierarchy of structure) is important for many biological materials’ properties. Dr. Laura Bagge studies invisibility in deep sea ocean life, and she presented how the size of the tiny microfibrils that make up larger muscle fibers can change how opaque an organism is — larger microfibrils have fewer interfaces for light to interact with, allowing the whole body of some shrimp species to be transparent.

These kinds of hierarchies are more commonly associated with strength, as in the example that Dr. Adam van Casteren presented. He studies how enamel, the outer layer of the tooth, resists wear, showing work suggesting that different levels of the material structure (nanostructure vs. microstructure) might respond differently to evolutionary pressure. That means that these hierarchies might have evolved to protect against damage from different types of diets, i.e. abrasion from sand particles in plant-based diets versus fracture from breaking apart bones and shellfish.

transparent-shrimp — Transparent shrimp achieve invisibility by having larger muscle microfibrils. (Image courtesy of Dr. Laura Bagge)

Microfluidics

Fluid transport (both liquids and gases) is crucial for organism survival, so it’s no surprise that many biomaterials have been optimized for this function. Dr. Anna-Christin Joel presented work on how lizard scales and certain spider silks use capillary forces to manipulate fluids. The same capillary control has been harnessed to transport water droplets collected along the body to the mouth for drinking and to make capture silk stick more tightly to prey (by pulling waxes up from the surface of insects).

In a different application of fluid handling, Prof. Cassie Stoddard talked about the large eggs of emus. All eggs have pores that provide airflow to the growing chick, but the pores in emu eggs are forked not straight. This might help solve the challenge of getting enough breathable air into large eggs without weakening the shell enough that it could be crushed by the adult (interestingly this feature is also seen in dinosaur eggs!).

2018-03-072018-04-14

Self-assembling silk lasers

Rings, spheres, and optical resonators self-assembled out of silk

Original paper: 3D coffee stains

When I first learned about the coffee ring effect I thought it was super cool, but it seemed like an open-and-shut case. Why do rings form where some liquids, like spilled coffee, are left to dry? Roughness on the table causes the liquid to spread imperfectly across the surface, pinning the edges of the droplet in place with a fixed diameter. Because the diameter of the droplet can’t change during evaporation, new liquid must flow from the droplet’s center to the edges. This flow also pushes dissolved coffee particles to the edges of the droplet, where they are left behind to form a ring as the water evaporates away (Figure 1). More details can be found in our previous post, here. It’s a complicated phenomenon, but after being described in 1997 it doesn’t seem like anything new would be going on here. Right? Well, as usually happens in science, classic concepts have a way of popping back up in unexpected ways. Last year It?r Bak?? Do?ru and her colleagues in Prof. Nizamo?lu’s group at Koç University, Turkey published a study using the often troublesome coffee ring effect to their advantage: making self-assembling silk lasers.

pinning — Figure 1: Pinning and the Coffee Ring Effect. A cross section of a water droplet drying on a smooth surface (A) versus a rough surface (B). On a smooth surface the droplet shrinks due to evaporation. On a rough surface the edge of the droplet is pinned and cannot shrink, forcing an internal flow to maintain the droplet’s shape.

The fundamentals here are the same as the classic coffee ring effect, but instead of coffee particles Do?ru’s droplets hold a colloidal suspension of silk fibroin proteins. In a colloidal suspension, particles (such as proteins) are mixed in another material (such as water) and neither dissolve fully into solution nor precipitate out. Smoke, milk, and jelly are all examples of colloids. Harnessing the coffee ring effect to build 2D structures out of colloidal particles has been well developed since Witten’s description of the coffee ring effect in 1997 [1], but 3D self-assembly is much less common. What makes Do?ru’s 3D structures possible is the fibroin protein.

Fibroin is the primary component of silk from the silkworm Bombyx mori. These fibers have been used by humans for thousands of years to make textiles, but recently the fibroin protein has taken on new life when extracted from silk as an aqueous, water-based, suspension and regenerated into other forms [2,3]. Fibroin proteins are long, and they easily tangle up and bond to each other to form networks of layered crystalline structures called beta-sheets (?-sheets) (Figure 2). These sheets give silk fibers and other fibroin materials strength and toughness. Furthermore, fibroin materials are biocompatible and biodegradable.

Silk Fibroin and Beta Sheets — Figure 2: Silk Fibroin And ?-sheets. Silk is made of long fibroin proteins (a) that have a repeating molecular structure. These proteins bond together into ?-sheets (b), which then stack together (c) to form materials with high strength and toughness.

To create 3D structures with the coffee ring effect, Do?ru, Nizamo?lu, and their coworkers put droplets of silk solution on superhydrophobic surfaces. Superhydrophobic surfaces strongly repel water, preventing water-based liquids from spreading flat across the surface and making the droplets stand straight up during the drying process. This makes the angle between the edge of the droplet and the surface (called the contact angle) particularly high, between 95-145 degrees throughout evaporation. The interaction between water and the superhydrophobic surface determines the shape of the final structure, with high contact angles creating more spherical droplets (Figure 3). After a solid 2D ring of fibroin forms on the bottom, the silk proteins continue to stack along the droplet’s surface, forming a stable spherical shell of ?-sheets that the remaining water can evaporate through. The researchers found that the concentration of the fibroin solution was important for controlling the final structure. If the solution is too dilute then the shell will collapse in on itself, but if the fibroin concentration is too high the initial contact angle will be lower and the final structure will also be more 2D than 3D.

To make 3D spheres, the researchers tried the pendant drop method, hanging a droplet from the tip of a needle. Similar to getting high contact angles from a droplet on a hydrophobic surface, hanging a droplet from a needle gives that droplet a small contact area, and a spherical shape (Figure 4). If the diameter of the needle is the same size or smaller than the contact area of the droplet on a superhydrophobic surface, then the shape of a droplet squeezed out of the needle should be as or more spherical than the droplets in the previous experiment. In this study, the pendant drop method ends up producing more uniform drying. These pendant-drop shells are smooth enough inside to act as optical resonators, surfaces that reflect light waves back on themselves so the waves amplify each other (the “a” in “laser,” which I always forget comes from the acronym for “light amplification by stimulated emission of radiation”).

As a proof of concept, the researchers made shells out of fibroin mixed with green fluorescent protein (GFP). Fibroin ?-sheet formation is so stable that it still happens when small amounts of other materials are present, so the optical resonator can form in the same way it did with a fibroin-only solution. In this case, because GFP has been added, when the structure is exposed to the right light source it will amplify green light emitted by the shell itself – an “all protein laser” in the making.

Figure 4: Benefits of the Hanging Pendant Drop. The hanging pendant drop method can produce similar spherical drops to a hydrophobic surface. It was shown that the pendant drop method produces more spherical final structures (adapted from Do?ru’s paper).

Part of what’s exciting about this publication is that the authors harness the coffee ring effect for a fun new type of small scale, self-directed 3D “printing.” They showed that the method works for other polymers as well, but I agree with their choice to highlight the silk protein fibroin. Not only is fibroin biocompatible, but it also has the potential to be more environmentally friendly to process than other polymers and is already produced in large quantities globally as part of the textile industry.

[1] Han, W. and Lin, Z. “Learning from ‘Coffee Rings’: Ordered Structures Enabled by Controlled Evaporative Self-Assembly.” Angew. Chem. Int. Ed. 51 (2012): 1534–1546.

[2] Altman, G.H. et al. “Silk-based biomaterials.” Biomaterials 24 (2003): 401–416.

[3] Koh, L.-D. et al. “Structures, mechanical properties and applications of silk fibroin materials.” Prog. Polym. Sci. 46 (2015): 86–110.