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Scientists at The University of Texas at Austin have developed a new microscopy technique for looking at nanoscale structures in biological samples that is analogous to using a glowing rubber ball to image a chair in a dark room Illustration by Jenna Luec

Researchers at The University of Texas at Austin (US) have demonstrated a new method for making three-dimensional images of structures in biological material, under natural conditions at a much higher resolution than achieved with previous methods. The method, called thermal noise imaging, may enable scientists to better understand how cells communicate with each another and to provide important insights for engineers developing artificial organs and tissue.

Taking crisp 3D images of nano-structures in soft biological samples is very difficult because of the so-called Brownian motion effect (tiny fluctuations in heat cause structures to wiggle). Other super-resolution imaging techniques often compensate for the resulting blurriness by stiffening various structures with chemicals, which has the huge drawback of making the materials lose their natural mechanical properties. Alternatively, focusing on rigid structures stuck to a glass surface limits the kinds of structures and configurations scientists can study.

A fresh approach

The UT Austin team, on the other hand, takes a very different approach: To make an image, they add nanospheres, which reflect laser light, to their biological samples under natural conditions, shine a laser on the sample and compile superfast snapshots of the nanospheres viewed through a light microscope.

Bouncing out the 3D image

Their analogy for thermal noise imaging is making a three-dimensional image of a room in total darkness by throwing a glowing rubber ball into the room and use a camera to collect a series of high-speed images of the ball as it bounces around. Bouncing around the room, the ball would be unable to move through solid objects such as tables and chairs. Combining millions of images taken so fast that they do not blur, one would be able to build a picture of where there are objects (wherever the ball could not go) and where there are no objects (where it could go).

In thermal noise imaging, the equivalent of the rubber ball is a nanosphere that moves around in a sample by natural Brownian motion – which now useful to the scientists, who are in fact taking advantage of that chaotic wiggling that makes everything blurry for most microscopy techniques.  

What is new and unique about thermal noise imaging

“Thermal noise imaging is a new way of looking at soft material at the nanometer scale,” says UT Austin physicist Ernst-Ludwig Florin. “Soft matter is defined by its mechanical properties and our microscope is specialized in feeling its softness with unmatched sensitivity and precision in three-dimensions. We use the touch sense to feel where objects are instead of eyes to look at them.” 

Observing mechanical properties of living cells

The expert notes that this is the first microscope that can do this in three-dimensions with nanometer scale resolution. However, the researchers still use light to detect the position of the probe particle, our touch sensor. The UT Texas team has demonstrated that thermal noise imaging works even under very difficult conditions, i.e., in samples that mess up the detector signal because they consist of many structures that change the light path. “Furthermore,” Florin adds, “we measured for the first time the tiny ‘vibrations’ of the filaments that make the network. These vibrations are important for the understanding of the mechanical properties of the network. The mechanical properties in turn are important for our understanding of macroscopic properties like skin elasticity, etc. So our thermal noise imaging microscope allows to look at soft matter, which includes living cells, from the most important angle, namely its mechanical properties.”

Advantages of thermal noise imaging over previous microscopy techniques

According to Florin, other super-resolution microscopes use fluorescent labels, little light bulbs that decorate the structures to be visualized, to find where these structure are. These light bulbs are dim and the microscopes have to integrate over a long time to localize them. The physicist likens these previous methods to taking photographs at night: They require long exposure times and, if objects move, the pictures are blurred. 

Waiting out higher resolution

“Since we use a very bright light source, a laser, to measure the position of the probe particle, our microscope does not have this problem to the same extent,” Florin says. “We are able to take fast snapshots with negligible motion blur.” The novel technique demonstrated by the UT Austin team also does not require attaching light bulbs to the image structures and the resolution does not depend on the density of these light bulbs. “We just have to sit there and wait until the particle explores the space. If we need higher resolution, we just wait a little longer.”

Florin notes that although the motion blur and the requirement for dense labeling of structures are important points, the more important point is that the new microscope goes beyond localizing matter. “It does not just answer the question where a certain structure is, but also how soft it is,” he says. “There is currently no other microscope that can do this in three-dimensions.”

The scientist agrees that his team’s pioneering thermal noise imaging method — and the ground-breaking findings it enables — could have significant impact on designing the next generation of light-based technologies, medicine, biology, and beyond: “Thermal noise imaging will help us to better understand the mechanical properties of soft structures in our body and allows for a rational design of novel material based on this understanding,” he says. “Collagen, for example, is used as a scaffold for building artificial skin, and understanding the inner workings of the network it forms from the perspective of a cell that grows in it might help us to design skin with properties closer to our natural skin.”

Outlook

Their scientific article, published in Nature Communications, established that light-based precision measurements on the nanometer scale are possible even under difficult conditions. “It is clear that this method can be improved in precision but also expanded in the information that can be extracted from the signals,” Florin says. “There is more to come. Stay tuned.”

In that regard, Florin briefly elaborates on the “many aspects” of what is next for him and his colleagues in this research endeavor. “In terms of technical improvements, we need to improve the stability of the microscope and improve scanning strategies so that we can image larger volumes.” The team aims to even better understand the physics behind it all, in an effort to improve what can be learned from thermal noise images.

“Finally, we plan to look at materials with different mechanical properties, in order to learn which information can be extracted from the images,” he says. “We are not short of problems to solve.”

Written by Sandra Henderson, research editor Novus Light Technologies Today

Labels: Thermal noise imaging,The University of Texas at Austin,3D imaging,3D microscopy,Brownian motion,super-resolution imaging,Ernst-Ludwig Florin

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