X-ray Microscopy and Imaging: Biology and Life Science

There are of numerous different applications of X-ray Microscopy and Imaging to the Life Sciences; the following pages only try to highlight a few examples that the reader may find of interest. The examples we show here range from the role of trace metals in single cells to the understanding of organismal form and function.

Understanding organismal form and function

Biomechanics is a discipline that uses principles of basic physics and engineering to understand how organisms function from a mechanical perspective.  Broadly speaking, one can ask how the laws of physics have shaped the history of life, and more specifically, how biomechanical relationships influence an organism's morphology, behavior, and ecology. This research has focused on three major lines of work, unified by a central question: What is the role of fluid mechanics in locomotion, breathing, and feeding?

How do insects breathe?

Insects breathe differently than vertebrate lung bellows system that most people are familiar with.  Instead, insects have a vast and interconnected system of tracheal tubes that run through every part of the body.  Respiratory gases (O 2 and CO 2 ) enter and leave this system through controllable valves called spiracles.  How do these gases move to and from the tissues?

It was once thought that diffusion alone should suffice, but researchers as early as the 19 th century have shown that some insects use movements of the body while breathing.  However, studies of insect respiration have generally focused on diffusion. In 2003, Mark Westneat, Wah-Keat Lee, and colleagues introduced a technique new to biology to visualize with unprecedented clarity the internal workings of small, living insects.  Taking advantage of extremely bright and coherent synchrotron x-rays at Argonne National Laboratory, they used phase-contrast imaging to observe previously unseen active cycles of inflation and deflation of tracheal tubes in multiple insect taxa.  (Click here to see a sample x-ray video; please note this will take you to a different domain).  Similar cycles were described early in the 20th century in a transparent flea, but because most insects are opaque, this phenomenon has gone unnoticed. Multiple lines of inquiry derived from this discovery are under investigation:

1. What is the role of convection to an insect's physiology?
2. How is convection produced, both from a morphological and a fluid mechanical perspective?
3. Why do some insects actively ventilate their tracheal systems, and others don't?
4. How important was convection to the evolution of insects, perhaps the most successful multicellular animals on earth?

This work has focused on the group of beetles represented in the figure, the ground beetles (Carabidae), but we have investigated a range of groups that include flies, grasshoppers, hemipterans, and earwigs.  Being able to visualize the tracheal system while the animals are alive is just one piece of the puzzle; we also use techniques such as the manipulation and measurement of respiratory gases.

How do insects ingest fluids during feeding?

Many insects ingest fluids, with sources ranging from nectar to blood to plant xylem and phloem. Such insects have tremendous economic importance for agriculture and human health worldwide.  However, our understanding of fluid ingestion in insects suffers a large gap due to our previous inability to see inside the animal while alive.

As with the mechanics of breathing in insects, synchrotron x-ray phase-contrast imaging has opened up a whole new vista into our understanding of fluid feeding.  Recently we have developed a technique to radiolabel food with elements such as iodine that become opaque when x-rays are tuned to the element's k-edge absorption energy.  In one example, we have found that the cabbage white butterfly, Pieris rapae , uses a two-stage suction/positive displacement mechanism in the cibarial pump, which drives flow through the head.  Food collects in the anterior esophagus before being moved posteriorly through the foregut in a larger, discrete bolus, contradicting previous models of butterfly feeding that assume a continuous flow of food through the system.  Our future work intends to fully test such models and to explore feeding diversity in lepidopterans. Questions of interest include:

1. How do pumps work to drive fluid flow?
2. Why do some insects use multiple pumps in the head?
3. What is the effect of food viscosity on fluid transport?
4. How does digestive system design reflect the type of food ingested?
5. Do species with more flexible designs better withstand environmental perturbations?

Questions ?

Jake Socha ph: 630.252.8548 ; email: jjsocha@aps.anl.gov

The roles of trace metals in cells

The presence of metals and trace elements is essential for the existence of life as we know it. In any organism, there are very few intracellular processes that are not dependent on the presence of metals or other trace elements. For example, it is estimated that one-third of all known proteins contain metal cofactors, and the majority of these function as essential metalloenzymes catalyzing biochemical reactions. Trace metals are increasingly recognized as having a critical impact on human health both in their natural occurrence and via therapeutic drugs (e.g., environmental exposure to heavy metals, treatment with cisplatin-based drugs in chemotherapy, and development of nanocomposites for gene therapy), and in diseases such as Alzheimer's. Quantitative study of the distribution of trace elements on the cellular and subcellular level provide important information about functions and pathways of metalloproteins and therapeutic approaches, especially in conjunction with the local chemical state of the elements of interest.

Hard x-ray fluorescence microscopy is a powerful technique to map and quantify element distributions in biological specimens such as cells and bacteria. It provides attogram sensitivity for transition metals like Cu, Zn, and other biologically relevant trace elements, combined with the capability to penetrate whole cells and thick tissue sections. Currently, a spatial resolution of 200 nm can be achieved routinely in mapping applications, and next-generation microprobes with a resolution limit of 30 nm are being planned, constructed, and tested [1]. The possibility of selecting the incident x-ray energy enables microspectroscopy and chemical state mapping to determine the speciation of elements of interest. These unique capabilities of x-ray fluorescence microscopy have been employed in diverse biomedical applications, and complement other modern microscopy techniques.

Elemental maps (55x30 microns in size) showing P, K, Ca, Fe, Cu, and Zn concentrations (in µg/cm 2 ) of a single rat cardiac myocyte. The nucleus of the cell is visible near the top of the cell in the center of the image as an area of increased Zn concentration. More strikingly, transverse striations are clearly visible in the Zn channel, with a regular periodicity of ~1.65 microns, corresponding to the periodicity of the sarcomere. Superposition of a light micrograph (not shown) on the x-ray fluorescence maps suggests that the transverse striations of high P, Fe, and Zn, and of low S coincide with the I-band of the intact cardiomyocyte [2].

Questions ?

Stefan Vogt - ph: 630.252.3071 ; email: vogt@aps.anl.gov