Every plant, animal and human is a wealthy microcosm of tiny, specialized cells. These cells are a world unto themselves, each with their very own unique parts and processes that transcend the naked eye. Viewing the inner workings of those microscopic constructing blocks at nanometer resolution without damaging their delicate organs has been a challenge, but scientists from various disciplines on the US Department of Energy's (DOE) Brookhaven National Laboratory have found an efficient method. . A cell using multiple techniques. The interesting means of obtaining these images was published.
Being in a position to understand the inner structure of cells, the way in which chemicals and proteins interact inside them, and the way these interactions dictate certain biological processes at nanometer resolution has led to advances in medicine, agriculture, and lots of other vital fields. can have significant effects. This work can also be paving the way in which for improved biological imaging techniques and recent devices to enhance biological imaging.
“Studying human cells and the organelles inside them is exciting,” said Qun Liu, a structural biologist on the Brookhaven lab, “but there are many opportunities to take advantage of our multimodal approach that combines hard X-ray computed tomography and X-rays. “Fluorescence imaging. We can study pathogenic fungi or helpful bacteria. We can see not only the structure of those microorganisms, but in addition the chemical processes when the cells interact in alternative ways.”
Removing certainly one of the constructing blocks of life
Before the researchers began imaging, their biggest challenge was preparing the sample itself. The team decided to make use of a cell from the human embryonic kidney (HEK) 293 line. These cells are known to be easy to grow but difficult to take multiple x-ray measurements. Although they're very small, cells are very sensitive to break attributable to X-rays.
The scientists went through a careful, multi-step process to make the sample stronger. They used paraformaldehyde to chemically preserve the cell structures, then a robot flash-frozen the samples by placing them in liquid ethane, transferring them to liquid nitrogen, and eventually removing the water however the cellular structures. Freeze-dried them to preserve them. Once the method was complete, the researchers placed the freeze-dried cells under a microscope and labeled them for targeted imaging.
At only 12-15 microns in diameter (the typical human hair is 150 microns thick), establishing the sample for measurement was hard, especially for measurements at different beamlines. The team needed to be sure that the cell structure could survive multiple measurements with high-energy X-rays without damage and that the cell might be reliably held in place for multiple measurements. Is. To overcome these obstacles, scientists developed standardized sample holders for use on multiple instruments and implemented optical microscopes to rapidly locate and image cells and minimize long exposures to X-rays. What can harm him?
Multimodal measurement
The team used two imaging techniques found on the National Synchrotron Light Source II (NSLS-II) – a DOE Office of Science user facility in Brookhaven – X-ray computed tomography (XCT) and X-ray fluorescence (XRF) microscopy.
The researchers collected XCT data, which uses X-rays to inform scientists a couple of cell's physical structure, on the Full-Field X-ray Imaging (FXI) beamline. Tomography uses X-rays to indicate a cross-section of a solid sample. A well-recognized example is a CT scan, which is utilized by medical professionals to create a cross-sectional image of any a part of the body.
The researchers collected XRF microscopy data, which offer further clues in regards to the distribution of chemical elements inside the cell, on the Submicron Resolution X-ray Spectroscopy (SRX) beamline. In this system, researchers direct high-energy X-rays at a sample, which excites the fabric and causes it to emit X-ray fluorescence. The X-ray emission has its own unique signature, which allows scientists to know what elements a sample accommodates and the way they're distributed to perform their biological functions.
“We were inspired to combine XCT and XRF imaging based on the unique, complementary information each provides,” said Xianghui Xiao, lead beamline scientist at FXI. “Fluorescence gives us a lot of useful information about trace elements inside cells and how they are distributed. This is very important information for biologists. Getting a high-resolution fluorescence map on many cells is very time consuming. It can be demanding, though. For a 2D image, it can take several hours.”
This is where obtaining a 3D image of the cell using XCT is useful. This information may also help guide fluorescence measurements at specific points of interest. This saves time for scientists, increases throughput, and likewise ensures that the sample doesn't should be exposed to X-rays for long periods of time, which may damage fragile cells. Potential damage could be minimized.
“This correlation approach provides useful, complementary information that can advance many practical applications,” commented Yang Yang, a beamline scientist at SRX. “For something like drug delivery, specific subsets of organelles can be identified, and then specific elements can be traced as they redistribute during treatment, giving us a clearer picture. How these drugs work at the cellular level.”
Although these advances in imaging have provided a greater view into the cellular world, there are still challenges to be faced and ways to further improve imaging. As a part of the NSLS-II Experimental Tools III project — a plan to construct recent beamlines to supply recent capabilities to the user community — Yang is the science lead of the team working on the upcoming Quantitative Cellular Tomography (QCT) beamline. , which will probably be dedicated to bioimaging. QCT is a full-field soft X-ray tomography beamline for imaging frozen cells with nanoscale resolution without the necessity for chemical fixation. This Cryosoft X-ray tomography beamline will complement existing methods, providing more detail into cellular structure and performance.
Future results
While having the ability to peer into the cells that make up the systems within the human body is exciting, having the ability to understand the pathogens that invade and disrupt these systems may also help scientists fight infectious disease. .
“This technology allows us to study the interaction between a pathogen and its host,” Liu explained. “We can look at the pathogen and a healthy cell before infection and then image them during and after infection. We will see structural changes in both the pathogen and the host and gain a better understanding of the process. “We also can study the interactions between helpful bacteria within the human microbiome which have a symbiotic relationship with plants.”
Liu is currently working with scientists from other national laboratories and universities for DOE's Biological and Environmental Research Program to check the molecular interactions between sorghum and the pathogenic fungus that causes anthracnose, a disease of plant leaves. can damage Sorghum is a crucial DOE bioenergy crop and the world's fifth most vital cereal crop, so understanding the tactics of this destructive fungus and the way sorghum defenses work on the cellular and molecular level has much to realize. Will be.
Being in a position to see at this scale can provide scientists insight into the wars being waged by pathogens on crops, the environment and even human bodies. This information may also help develop the precise tools to fight these attackers or fix systems that aren't working optimally at a basic level. The first step is having the ability to see a world that the human eye can't see, and advances in synchrotron science have proven a strong tool in uncovering it.
This work was supported by Laboratory Directed Research and Development funding from Brookhaven and the DOE Office of Science.