Anyone who has used a microscope in a biology class to study a plant cell will probably be able to recall a similar situation. It is easy to tell that a certain chloroplast is located above and to the right of the nucleus. But are both of them located on the same plane? Once you adjust the focus on the microscope, however, you see that the image of the nucleus becomes sharper while the image of the chloroplast blurs. One of them must be a little higher and one a little lower than the other. However, this method cannot give us precise details about their vertical positions.

The principle is very similar if you want to observe individual atoms instead of cells. So-called quantum gas microscopy can be used for this purpose. It allows you to straightforwardly determine the x and y coordinates of an atom. However, it is much more difficult to measure its z coordinate, i.e., the distance to the objective lens: In order to find out on what plane the atom is located, multiple images must be taken in which the focus is shifted across various different planes. This is a complex and time-consuming process.

Turning round specks into dumbbells

“We have now developed a method in which this process can be completed in one step,” explains Tangi Legrand from the Institute of Applied Physics (IAP) at the University of Bonn. “To achieve this, we use an effect that has already been known in theory since the 1990s but which had not yet been used in a quantum gas microscope.”

To experiment on the atoms, it is first necessary to cool them down significantly so that they are barely moving. Afterwards, it is possible, for example, to trap them in a standing wave of laser light. They then slip into the troughs of the wave similar to how eggs sit in an egg box. Once trapped, to reveal their position, they are exposed to an additional laser beam, which stimulates them to emit light. The resulting fluorescence shows up in the quantum gas microscope as a slightly blurred, round speck.

“We have now developed a special method to deform the wavefront of the light being emitted by the atom,” explains Dr. Andrea Alberti. The researcher, who has now moved from the IAP to the Max Planck Institute of Quantum Optics in Garching, also participated in the study. “Instead of the typical round specks, the deformed wavefront produces a dumbbell shape on the camera that rotates around itself. The direction in which this dumbbell points is dependent on the distance that the light had to travel from the atom to the camera.”

“The dumbbell thus acts a bit like the needle on a compass, allowing us to read off the z coordinate according to its orientation,” says Prof. Dr. Dieter Meschede. The IAP researcher, whose research group carried out the study, is also a member of the transdisciplinary research area “Matter” at the University of Bonn.

Important for quantum mechanics experiments

The new method makes it possible to precisely determine the position of an atom in three dimensions with one single image. This is important, for example, if you want to carry out quantum mechanics experiments with atoms because it is often essential to be able to precisely control or track their position. This allows researchers to make the atoms interact with one another in the desired way.

Furthermore, the method could also be used to help develop new quantum materials with special characteristics. “For example, we could investigate which quantum mechanical effects occur when atoms are arranged in a certain order,” explains Dr. Carrie Weidner from the University of Bristol. “This would allow us to simulate the properties of three-dimensional materials to some extent without having to synthesize them.”

The study, led by Professor Jun Suzuki of the Institute for Integrated Cell-Material Sciences (iCeMS), addresses a critical gap by directly targeting genes within testicular cells inside living organisms. Utilizing a genetic tool called CRISPR, which can be likened to genetic scissors, the researchers developed a method to investigate which genes contribute to healthy sperm production in living animals. Until now, this had mainly been done in cells cultivated in the laboratory. The researchers randomly disrupted genes by developing a method to introduce a collection of genetic tools in lentivirus into testicular cells in testes at high efficiency. Through this method, the researchers made it possible to analyze the effects of targeted genes on specific biochemical reactions in the sperm cells, such as the movement of lipids (fats) in cell membranes.

Using the method, the team focused on sperms with defective capacitation, a process through which sperms become capable of fertilizing an egg. They identified these sperms by measuring how much calcium they absorbed. By using this method in live animals, they were able to identify a specific gene, Rd3, as being crucial for maintaining sperm cell health, particularly during sperm cell development. Despite Rd3’s previous association with eye function, the team found it is also highly active in round sperm cells, an early stage in sperm production, and that it plays a significant role in regulating sperm health. This discovery was made possible by examining how Rd3 interacts with mitochondria, structures responsible for energy production within cells.

To further understand Rd3’s function, Suzuki and his colleagues developed Hub-Explorer, a computational tool that revealed Rd3’s impact on regulation of oxidative stress — a condition linked to cellular damage. Rd3’s role in regulating oxidative stress revealed its significance in maintaining sperm integrity during development.

“While women have many available birth control options, choices for men remain limited,” says Noguchi, a first author of this paper. “This new screening method holds promise by aiding the discovery of key molecules, potentially leading to new birth control options and infertility therapies for men.”

“These discoveries not only advance our understanding of sperm cell development but also demonstrate the potential to uncover mysteries in different biological processes,” adds Suzuki. “The method can also be applied to other tissues, potentially expediting the development of drugs for a wide range of diseases.”

Despite these advancements, challenges remain. The researchers observed a gradual decline in the number of cells with targeted genes, suggesting room for improvement. They are currently exploring alternative approaches to enhance the effectiveness of their technique, and they plan to apply it to investigate various biological processes and diseases in other areas of the body.

Findings of the study led by Gaurav Sahay and Yulia Eygeris of the Oregon State University College of Pharmacy and Renee Ryals of Oregon Health & Science University were published today in the Proceedings of the National Academy of Sciences.

Unlike other types of lipid nanoparticles that tend to accumulate in the liver, the ones in this study, based on the compound thiophene, are able to navigate their way to the tissues of the lungs and retina, where they deliver their therapeutic payload. The researchers refer to these new lipids as Thio-lipids.

The collaboration demonstrated, by using animal models, the possibility of using Thio-lipids in lipid nanoparticles to deliver messenger RNA, the technology underpinning COVID-19 vaccines, to combat genetic blindness and pulmonary disease.

“These nanoparticles filled with fatty lipids can encapsulate genetic medicines like mRNA and CRISPR-Cas9 gene editors, which can be used to treat and even cure rare genetic diseases,” said Eygeris, a senior research associate at OSU. “Chemical structures of the lipids determine how potent are the lipid nanoparticles and which organ they can reach from the bloodstream.”

Lipids are organic compounds containing fatty tails and are found in many natural oils and waxes, and nanoparticles are tiny pieces of material ranging in size from one- to 100-billionths of a meter. Messenger RNA delivers instructions to cells for making a particular protein.

With the coronavirus vaccines, the mRNA carried by the lipid nanoparticles instructs cells to make a harmless piece of the virus’ spike protein, which triggers an immune response from the body.

As a therapy for vision impairment resulting from inherited retinal degeneration, the mRNA would instruct cells in the retina — which don’t work right because of a genetic mutation — to manufacture the proteins needed for sight. Inherited retinal degeneration, commonly abbreviated to IRD, encompasses a group of disorders of varying severity and prevalence that affect one out of every few thousand people worldwide.

An example of a genetic pulmonary condition is cystic fibrosis, a progressive disorder that results in persistent lung infection and affects 30,000 people in the U.S., with about 1,000 new cases identified every year.

One faulty gene — the cystic fibrosis transmembrane conductance regulator, or CFTR — causes the disease, which is characterized by lung dehydration and mucus buildup that blocks the airway.

The thiophene-based lipid nanoparticle study, which involved mice and non-human primates, stems from a $3.2 million grant to Sahay and Ryals from the National Eye Institute. The grant’s purpose is addressing limitations associated with the current primary means of delivery for gene editing: a type of virus known as adeno-associated virus, or AAV.

“AAV has limited packaging capacity compared to lipid nanoparticles and it can prompt an immune system response,” said Sahay, a professor of pharmaceutical sciences. “It also doesn’t do fantastically well in continuing to express the enzymes the editing tool uses as molecular scissors to make cuts in the DNA to be edited.”

Sahay calls the Thio-lipid findings “highly encouraging” but says more studies are needed, including research on the lipids’ long-term impact on retinal health.

“But we think our results serve as a proof of concept and we will continue to explore Thio-lipids in potential treatments of pulmonary and retinal genetic diseases,” he said.

In addition to the National Eye Institute, funding and research support were provided by the Oregon National Primate Research Center and the Casey Eye Institute.