Now, EMBL scientists have combined artificial intelligence (AI) algorithms with two cutting-edge microscopy techniques — an advance that shortens the time for image processing from days to mere seconds, while ensuring that the resulting images are crisp and accurate. The findings are published in Nature Methods.

“Ultimately, we were able to take ‘the best of both worlds’ in this approach,” says Nils Wagner, one of the paper’s two lead authors and now a PhD student at the Technical University of Munich. “AI enabled us to combine different microscopy techniques, so that we could image as fast as light-field microscopy allows and get close to the image resolution of light-sheet microscopy.”

Although light-sheet microscopy and light-field microscopy sound similar, these techniques have different advantages and challenges. Light-field microscopy captures large 3D images that allow researchers to track and measure remarkably fine movements, such as a fish larva’s beating heart, at very high speeds. But this technique produces massive amounts of data, which can take days to process, and the final images usually lack resolution.

Light-sheet microscopy homes in on a single 2D plane of a given sample at one time, so researchers can image samples at higher resolution. Compared with light-field microscopy, light-sheet microscopy produces images that are quicker to process, but the data are not as comprehensive, since they only capture information from a single 2D plane at a time.

To take advantage of the benefits of each technique, EMBL researchers developed an approach that uses light-field microscopy to image large 3D samples and light-sheet microscopy to train the AI algorithms, which then create an accurate 3D picture of the sample.

“If you build algorithms that produce an image, you need to check that these algorithms are constructing the right image,” explains Anna Kreshuk, the EMBL group leader whose team brought machine learning expertise to the project. In the new study, the researchers used light-sheet microscopy to make sure the AI algorithms were working, Anna says. “This makes our research stand out from what has been done in the past.”

Robert Prevedel, the EMBL group leader whose group contributed the novel hybrid microscopy platform, notes that the real bottleneck in building better microscopes often isn’t optics technology, but computation. That’s why, back in 2018, he and Anna decided to join forces. “Our method will be really key for people who want to study how brains compute. Our method can image an entire brain of a fish larva, in real time,” Robert says.

He and Anna say this approach could potentially be modified to work with different types of microscopes too, eventually allowing biologists to look at dozens of different specimens and see much more, much faster. For example, it could help to find genes that are involved in heart development, or could measure the activity of thousands of neurons at the same time.

Next, the researchers plan to explore whether the method can be applied to larger species, including mammals.

A paper describing the study, led by Penn State Professor of Biology Hong Ma, appears in the May 2021 issue of the journal Molecular Plant.

“Legumes make up the third largest family of flowering plants and are incredibly diverse — ranging from tiny herbs to giant trees,” said Ma, who is the Huck Distinguished Research Professor of Plant Molecular Biology at Penn State. “They are essential food crops for both humans and livestock, can be used as lumber, and have many other uses. Maybe most importantly, they can ‘fix’ nitrogen — extracting the vital nutrient from the atmosphere and storing it in nodules on their roots in a symbiotic relationship with soil bacteria — making them important as green manure to improve soil health.”

There are over 19,000 species in the legume family divided into six subfamilies and then further divided into narrower and narrower groupings based on their evolutionary relationships. There are 765 genera — the grouping one level above species — of which the team sampled members of 333. To build the family tree, the team analyzed gene sequences from the transcriptomes — the portion of the genome that is expressed as genes — of most of the 463 species and a small number of shallowly sequenced whole genomes from across legume diversity.

“This is the largest study of this kind for a single plant family,” said Ma. “We went to great lengths to sample as many species as we could to get a broad representation of the legume family, but it is often difficult to get well-preserved specimens that we can extract DNA or RNA from, especially for species found in remote locations. Having this broad representation of species allowed us to build the most detailed nuclear-gene family tree for legumes to date.”

In addition to helping researchers understand the evolution and diversification of legumes, the new legume family tree helps to clarify the relationship between crop plants and their wild relatives. Although the close relatives of important agricultural crops are often known, studying more distant wild cousins could reveal traits that could be exploited to help plants thrive in changing environments and resist diseases or insect pests.

Across the legume family tree, the research team identified strong evidence for 28 separate whole-genome duplication events. Whole-genome duplications, evolutionary events that result in complete duplication of the entire genome, are fairly common among flowering plants and are thought to allow for functional innovation and evolutionary diversification. One of the duplication events that the team identified appears to have occurred in the ancestor of all members of the legume family.

“Because for most of the species in our study we used transcriptomes and do not have entire genome sequences, we consider these as ‘proposed’ genome duplication events,” said Ma. “These kinds of studies are kind of like solving a mystery. If you only have one or a few witnesses it might be difficult to convince a jury of your evidence, but if you have a hundred witnesses who have different perspectives and they all point to the same thing it becomes difficult to dismiss that evidence. In our case, the different species are like our witnesses. The size of our study allowed us to identify events that we might otherwise have dismissed.”

The two largest subfamilies account for over 17,000 legume species and include all of the species with the ability to fix nitrogen. Nitrogen is an important plant nutrient — most commercial fertilizers contain a mix of nitrogen, phosphorus and potassium — so the symbiotic relationship between some legumes and the microorganisms that allow them to assimilate nitrogen from the atmosphere using root nodules has spurred their success by allowing them to colonize areas with less fertile soil. The research team also identified clues to the evolution of the genes responsible for this important trait.

“Our data support the idea that nodulation and nitrogen fixation originated a single time early in the history of legumes and other related nitrogen-fixing plants and the whole-genome duplication event at the origin of legumes might have been crucial for the evolution of this process,” said Ma. “In addition to this duplication event, we are also able to see gene loss in plants that do not have the ability to nodulate, and evolutionary changes in genes that contributed to their role in nodulation.”

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Materials provided by Penn State. Original written by Sam Sholtis. Note: Content may be edited for style and length.

In their new analysis, published in the Journal of Clinical Sleep Medicine, the researchers found the number of medical visits and prescriptions filled were nearly doubled in people with sleep disorders such as sleep apnea and insomnia, compared to similar people without. Affected patients were also more likely to visit the emergency department and have more comorbid medical conditions.

Costly medical care for sleep disorder patients

The researchers sought out to determine the true diagnostic prevalence of sleep disorders and how expensive these conditions were to the health care system. They examined differences in health expenditures in similar patients with and without a sleep disorder diagnosis, as determined by their ICD-10 diagnosis code. The study included data from a nationally-representative survey of more than 22,000 Americans called the 2018 Medical Expenditure Panel Survey, which is administered by the Department of Health and Human Services’ Agency for Healthcare Research and Quality.

They found 5.6 percent of respondents had at least one sleep disorder, which translated to an estimated 13.6 million U.S. adults. This likely represents a significant underestimate, according to the authors, as insomnia alone is felt to conservatively affect 10 to 20 percent of the population. These individuals accumulated approximately $7,000 more in overall health care expenses per year compared to those without a sleep disorder — about 60 percent more in annual costs. This equates to a conservative estimate of $94.9 billion in health care costs per year attributable to sleep disorders.

The analysis revealed that patients with sleep disorders attended more than 16 office visits and nearly 40 medication prescriptions per year, compared to nearly 9 visits and 22 prescriptions for those without a sleep disorder. The study did not quantify non-health care related costs, but the authors noted it can be assumed that more doctors’ appointments means more time off from work, school or other social obligations, not to mention decreased productivity associated with symptoms, only exacerbating costs to society.

Sleep disorders raise risk for other conditions

Sleep disorders can take a toll on health and quality of life in numerous ways. Individuals with certain sleep disorders experience decrease daytime functionality related to sleepiness, mental fog and an increased risk of motor vehicle accidents, for instance. Obstructive sleep apnea is one of the most common sleep disorders and if untreated, can increase risk for neurocognitive issues, such as difficulty concentrating and mood disorders, as well as cardiovascular conditions including heart attacks, strokes, high blood pressure and irregular heart rhythms.

Getting a proper diagnosis at the sign of asleep problem can lead to an effective treatment for a sleep disorder.

“Fortunately, studies have demonstrated that treating certain sleep disorders effectively reduces health care utilization and costs. Therefore, sleep issues should not be ignored. Greater recognition of sleep disorders and an early referral to a sleep specialist are essential,” said Dr. Huyett. “Your sleep is important, and if there’s an issue with your sleep, seek help for it.”

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The new species, named Ampelorhiza heteroxylon, belongs to a diverse group of tropical lianas called Paullinieae, within the soapberry family (Sapindaceae). More than 475 species of Paullinieae live in the tropics today.

Researchers identified the species from fossilized roots that revealed features known to be unique to the wood of modern climbing vines, adaptations that allow them to twist, grow and climb.

The study, “Climbing Since the Early Miocene: The Fossil Record of Paullinieae (Sapindaceae),” was published April 7 in the journal PLOS ONE.

“This is evidence that lianas have been creating unusual wood, even in their roots, as far back as 18 million years ago,” said wood anatomist Joyce Chery ’13, assistant research professor in the School of Integrative Plant Science, Plant Biology Section, in the College of Agriculture and Life Sciences, and a corresponding author of the paper.

“Before this discovery, we knew almost nothing about when or where these lianas evolved or how rapidly they diversified,” said first author Nathan Jud, assistant professor of plant biology at William Jewell College and a former Cornell postdoctoral researcher.

Panama was a peninsula 18.5 to 19 million years ago, a volcanic landscape covered with tropical forest in North America and separated from South America by a Central American seaway. While these forests contained North American animals, the plants mostly descended from South American tropical plants that had dispersed across the seaway, Jud said.

“The fossil we described is the oldest macrofossil of these vines,” he said, “and they were among the plants that made it to North America long before the Great American Biotic Interchange when large animals moved between the continents some 3 million years ago.”

In the study, the researchers made thin slices of the fossil, examined the arrangements and dimensions of tissues and water conducting vessels under a microscope and created a database of all the features. They then studied the literature to see how these features matched up with the living and fossil records of plants.

“We were able to say, it really does look like it’s a fossil from the Paullinieae group, given the anatomical characteristics that are similar to species that live today,” Chery said.

During their analyses, the researchers identified features that are characteristic of lianas. Most trees and shrubs have water-conducting tissues (which transport water and minerals from roots to leaves) that are all roughly the same size when viewed in a cross-section; in vines, these conduits come in two sizes, big and small, which is exactly what the researchers discovered in the fossil.

“This is a feature that is pretty specific to vines across all sorts of families,” Chery said.

The two vessel sizes provide insurance for a twisting and curving plant, as large vessels provide ample water flow, but are also vulnerable to collapse and develop cavities that disrupt flow. The series of smaller vessels offers a less vulnerable backup water transport system, Chery said.

Also, cross-sections of the wood in trees and shrubs are circular, but in the fossil, and in many living vines, such cross-sections are instead irregular and lobed.

Thirdly, on the walls of those vascular vessels, they found long horizontal perforations that allow for water to flow in lateral directions. That is a distinguishing feature of lianas in the soapberry family, Chery said.

In future work, now that they can place the lianas of Sapindaceae to 18.5 million years ago, the researchers intend to continue their investigation of the evolutionary history and diversification of this family. Chery also plans to investigate how wood has evolved in this group of vines, including identifying the genes that contribute to lobe-shaped stems.

The study was partly funded by the National Science Foundation.