3-D Cellular Ultrastructure Can Be Resolved by X-ray Microscopy
This shows X-ray images of mouse cancer cells. 3-D partially-coherent X-ray tomograms of mouse adenocarcinoma cells reveal numerous sub-cellular organelles including mitochondria (M), lyosomes (L), endoplasmic reticulum (ER), vesicles (V), the plasma membrane (PM), the nuclear membrane (NM), nuclear pores (NP), nucleoli (Nu) and nuclear membrane channels (NMC). Also visible are sub-organellar structures such as cristae in the mitochondria and dense inclusions in the lysosomes. All images were acquired with a 25 nm zone plate at 510 eV photon energy, except for (b) which was acquired with a 40 nm zone plate. xy (a,b,e,f) and xz (c,d) slices of the 3D tomograms are shown. Pixel sizes and slice thicknesses are 9.8 nm for (a,e,f) and 15.6 nm for (b). Scale bars ≡ 0.39 µm. (Photo credit:G. Schneider, J. McNally for Nature Methods)
X-ray microscopy (XRM) is more rapid than cryoelectron tomography or super-resolution fluorescence microscopy and could fill an important gap in current technologies used to investigate in situ three-dimensional structure of cells. New XRM methods developed by first author Gerd Schneider, Ph.D., working with James McNally. Ph.D., and a team of colleagues, is capable of revealing full cellular ultrastructure without requiring fixation, staining, or sectioning.
In a recent paper in Nature Methods, the team of scientists describe the first demonstration, to their knowledge, that X-ray tomography can surpass the resolution of conventional light microscopy. To construct their X-ray microscope, the researchers combined a high resolution X-ray objective with partially coherent illumination from a synchrotron radiation source, which left room for a flat sample holder for cells that was capable of tilting ±79 degrees. This design resulted in better contrast of the cell’s fine features and allowed for the examination of adherent cells.
The researchers used their X-ray microscope to image intact mouse adenocarcinoma cells grown on coated sample grids and vitrified in liquid ethane. Images were acquired at tilt angles from ±60 degrees in increments of one degree. Total X-ray exposure produced negligible damage. The authors processed the images using a reciprocal space algorithm to generate tomograms of fourteen cells. The images included subcellular features either undetected or faintly detected by previous XRM studies and exhibited good contrast of membrane-bound structures, including those of internal organelles. The researchers were not, however, able to detect ribosomes or the mitochondrial double membrane likely because they are below the current detection limit and because the restricted tilt angle led to poorer z-dimension resolution.
To determine the resolution of their new X-ray method, the authors used two different measures: the Rayleigh criterion, comparable to super-resolution fluorescence microscopy, and the Fourier ring correlation, comparable to cryoelectron tomography. Using the Rayleigh criterion, the images had a resolution of 36nm. Using the Fourier ring correlation, the tomograms had a best resolution of 70nm (equivalent to 35nm half-pitch resolution).
Future improvements to this XRM method may be achieved by combining improved X-ray optics with super-resolution fluorescence microscopy. This new method allows for a broad application to any sample capable of growing or being placed on a flat holder.Summary Posted: 11/2010
Gerd Schneider, Peter Guttmann, Stefan Heim, Stefan Rehbein, Florian Mueller, Kunio Nagashima, J. Bernard Heymann, Waltraud G. Muller, and James G. McNally Nat. Methods Epub 14 November 2010 http://www.nature.com/nmeth/journal/v7/n12/full/nmeth.1533.html