X-Ray Microscopy: Versatile Tool for Life Sciences Imaging

With a resolution that’s typically between that of optical and electron microscopy, X-ray microscopy (XRM) is a versatile tool for imaging intact hydrated cellular and subcellular structures (Cao et al., 2024). The impact of XRM is perhaps best evidenced by its global market size, which reached over $1.7 billion in 2023 and is estimated to reach over $3.6 billion by 2031; many envisioned XRM applications focus on biological imaging (Market Research Future, 2024).

What are the basic principles and utility of XRM? What are its advantages and disadvantages? What challenges are holding back the XRM field, and what is being done about that? Here, we provide an overview and representative commercial solutions, as well as recent research advances in method and tool development.

Basic principles and utility

XRM makes use of the water transparency window. This is simply the electromagnetic wavelength range (ca. 2.3–4.4 nm) over which oxygen—and thus water—only weakly absorbs X-rays. Thus, membranes and other carbon-rich regions are in high contrast to vacuoles and other dilute aqueous compartments. Historically, soft XRM (low energy;<5 keV) has used a synchrotron radiation source to image a cryogenically cooled sample. This radiation source is from particle accelerators that can cost tens to hundreds of millions of U.S. dollars to construct and are generally only available at major universities and national labs. Laser-based X-ray sources are a modern alternative that has increased the accessibility of XRM.

Philipp Bastians, Sales Manager at Zeiss, Life Science Electron & X-Ray Microscopy, North America, presents the need for XRM in life sciences research: “Biological specimens like bones, plants, and entire organisms are challenging samples for widely used light microscopy techniques. Alternatively, observation at high resolution requires elaborate and typically destructive preparation methods, undermining the integrity of the specimen and the features of interest. XRM allows for non-destructive imaging at high resolution and maintains the specimen’s integrity and context.”

Mouse embryo imaged with ZEISS Xradia Versa XRM to show internal structures. Single projection images on the left hand side and a 3D render shown on the right. Sample courtesy of Dr Zheng Zhifa, Beijing Union Medical College Hospitals.

Jolien Dellafaille, Application Scientist at Bruker, highlights representative life sciences applications of their XRM instruments: “The imaging possibilities are endless. You can scan coffee beans to evaluate how the internal structure changes upon roasting. You can also just as easily see how insects adapt to internal plant tissue. XRM gives gorgeous portraits of insects; there’s a lot for entomologists to learn.”

Advantages and disadvantages

XRM has many advantages for life science research (Loconte et al., 2023). An appropriate optical design can allow users to rapidly shift between imaging small and large cells. As in fluorescence microscopy, XRM is compatible with multi-parameter imaging. Additionally, sectioning is unnecessary for samples no more than ca. 10- to 20-µm thick. As in electron microscopy, XRM can have isotropic resolution and does not require—yet can use—contrast labeling.

Dellafaille says that Bruker’s XRM technology is straightforward and fast: “With our X-ray microscopes, you can clearly discern detailed alveolar structure after less than 24 hours of image acquisition, which might otherwise take around two weeks of preparatory work for conventional microscopy. This means it can also be used as a screening method to target sections of interest, saving time in preparation for other imaging techniques. Furthermore, the level of required software training can meet the needs of entry-level and advanced users.”

Mouse lung, stained and embedded, scanned on Bruker's SkyScan 2214CMOS, sample courtesy of Charité, Berlin 

Joe Mowery, Product Applications and Sales Specialist at Zeiss, Life Science Electron & X-Ray Microscopy, North America, says that their XRM technology is particularly beneficial when combined with volume electron microscopy (vEM): “vEM can image biological structures at nanometer resolution in three dimensions. However, vEM is destructive and time-consuming, and the acquired volume is often limited to a fraction of the specimen’s volume. Once the specimen is prepared for vEM acquisition it becomes opaque to conventional light microscopes, which makes it difficult to exactly pinpoint the volume of interest within the context of the prepared specimen. X-ray tomography harnesses the penetrative capability of X-rays to generate 3D datasets without physically cutting the specimen, and provides a detailed map for the consecutive vEM experiment.”

However, XRM also has two main disadvantages. One, flash-freezing techniques that are common in XRM are incompatible with the live-cell imaging enabled by fluorescence microscopy. Two, the commonly reported maximum resolution of soft XRM, ca. 25 nm (Loconte et al., 2023), is 10–100 times inferior to that of electron microscopy.

Challenges

Dellafaille presents a common challenge of XRM—lack of contrast—and how Bruker’s instruments solve this problem: “Bone scanning is easy; you often don’t need contrast agents to get all the information you need. Soft tissue—such as the lungs, kidneys, heart, and embryos—is more difficult to image and often requires a contrast agent. Alternatively, single distance phase retrieval reconstruction, offered with our X-ray microscopes, can substantially ease image interpretation without requiring a contrast agent. Compared with ex vivo imaging, in vivo imaging is more resolution-limited to avoid radiation damage, but is invaluable for measurements such as tidal lung volume, bone morphometry, BMD, and fat analysis.”

Steven Hernandez, Product Applications and Sales Specialist at Zeiss, Life Science Electron & X-Ray Microscopy, North America, highlights how their microscopes overcome the common challenge of restricted resolution at high magnifications: “While conventional micro-CT/X-ray imaging microscopes are resolution-limited due to the specimen dimensions—e.g., a smaller distance between the X-ray source and the specimen center corresponds to increased magnification—Zeiss offers an optical magnification technology called Resolution at a Distance (RaaD) that overcomes this resolution obstacle, even for larger samples. RaaD allows researchers to zoom into their specimens without the need to cut and extract the tissue, which might alter the very structure of interest. This RaaD capability is useful in fields like plant science, neuroscience, evolutionary biology, comparative anatomy, and pathology.”

Ongoing research efforts

Contrast labeling can aid interpretation of the biology indicated by XRM (Wang et al., 2024). Unfortunately, currently available probes often result in imprecise labeling, such as that arising from antigen–antibody crosstalk. Tang et al. (2024) devised a solution to this problem: clickable X-ray nanoprobes, which use labeling chemistry that doesn’t interfere with endogenous biology. Contrast labeling of HeLa cells was straightforward, such as for differentiating the Ni-labeled cell membrane from the Co-labeled nucleus.

What does the future hold for XRM? For example, it will be useful for identifying novel pancreatic ductal adenocarcinoma biomarkers from diagnostically relevant tissue structures (Pinkert–Leetsch et al., 2023). With its ability to image an entire functional cell, it is well-positioned as an experimental tool for developing and testing whole-cell simulations (Loconte et al., 2023). Integrating XRM with correlative light–electron microscopy will be invaluable for probing far-from-equilibrium biological events on the millisecond scale (Szabo and Burg, 2024). Furthermore, artificial intelligence will be useful for extracting ultrastructure detail from a sea of XRM data (Dyhr et al., 2023).

It is critical to understand the corresponding science to determine whether and how XRM can meet your laboratory’s needs. Speak with an industry specialist to avoid common pitfalls and make appropriate choices tailored to your application.

References

Cao M, et al. (2024). In situ label-free X-ray imaging for visualizing the localization of nanomedicines and subcellular architecture in intact single cells. Nat. Protoc. 19(1):30–59.

Dyhr MCA, et al. (2023). 3D surface reconstruction of cellular cryo-soft X-ray microscopy tomograms using semisupervised deep learning. Proc. Natl. Acad. Sci. USA 120(24):e2209938120.

Loconte V, et al. (2023). Soft X-ray tomograms provide a structural basis for whole-cell modeling. FASEB J. 37(1):e22681.

Market Research Future (2024). High-resolution 3D X-ray microscopy market research report information by type (sub-micron XRM and nanoscale XRM), by application (advanced package development, mineralogy discrimination, failure analysis and surface measurements), by end user (oil & gas, material science, semiconductor, metrology, life science and healthcare) - Forecast till 2032. Rep. ID MRFR/MED/0902-CR, Pune India.

Pinkert–Leetsch D, et al. (2023). Three-dimensional analysis of human pancreatic cancer specimens by phase-contrast based X-ray tomography – The next dimension of diagnosis. Cancer Imaging 23:43.

Szabo GV and Burg TP (2024). Time resolved cryo-correlative light and electron microscopy. Adv. Funct. Mater. Published ahead of print, Apr 16, 2313705.

Tang Q, et al. (2024). Clickable X-ray nanoprobes for nanoscopic bioimaging of cellular structures. JACS Au 4(3):893–902.

Wang S, et al. (2024). Emerging synchrotron radiation X-ray-sensitive probes for in situ bioimaging at the nanoscale. Trends Anal. Chem. 170:117453.