Advanced Light Source, 1 Cyclotron Road, Berkeley, CA 94720


Mesocale Cell imaging

Soft x-ray tomography can image an entire cell in its natural hydration state. Determining the location of specific molecular interactions requires the molecular participants are labeled with a marker that is visible either in the soft x-ray microscope or in another modality and the data correlated. The latter is the preferred option since direct visualization of a protein using soft x-rays requires it to be labeled with a heavy metal tag (a potentially damaging process to the integrity of the cell structure and molecular organization). Consequently, we have focused our technology development on correlated microscopy, in particular correlated cryogenic confocal microscopy. This technique is described in the below manuscript, followed by a recent research application. You can download a pdf version of these papers here and here)

Mark Le Gros and Axel Ekaman working on the Structured Illumination Cryo-Light Microscope

Soft X-ray microscopes have existed for many years, but it is only relatively recently that they have been successfully applied to imaging the cellular mesoscale (Weiß et al. 2000Denbeaux et al. 2001Meyer-Ilse et al. 2001Schneider et al. 2002). The reasons for this change are twofold. Firstly, a combination of technical advances – specifically, in the areas of detectors, X-ray sources, and optics – made soft X-ray microscopes capable of imaging cells more rapidly, and with higher spatial resolution. Secondly, cryogenic specimen rotation stages became available on soft X-ray microscopes, making tomographic data collection possible (Weiß et al. 2000Larabell and Le Gros, 2004a,b). Adopting a tomographic approach is essential for imaging the mesoscale organisation using a soft X-ray microscope. Without it, the microscope can only generate 2D projection images of the cell. In these projections, internal structures are superimposed on each other, making interpretation of the images virtually impossible (McDermott et al. 2012a,b).

SXT of a fibrosarcoma cell. (A) An orthoslice through a SXT reconstruction of a HT1080 fibrosarcoma cell. Image is comprised of tomographic reconstructions from two adjacent fields computationally stitched together. (B) 3D volume rendering based on organelles’ linear absorption coefficient (LAC) and morphology. Red: Nucleus, Turquoise: Mitochondria, Blue: Lipids, Gray: cell membrane. Scale bar = 1.5 µm.