5830 X-Ray Microscopy and the View Inside Living Cells

Friday, February 17, 2012: 10:30 AM
Room 208-209 (VCC West Building)
Carolyn Larabell , Lawrence Berkeley National Laboratory, Berkeley, CA
Soft X-ray Tomography (SXT) is similar in concept to the well-established medical diagnostic technique computed axial tomography (CAT), except SXT is capable of imaging with a spatial resolution of 50 nm, or better. In SXT, cells are imaged using photons with energies between the K shell absorption edges of carbon (284 eV, λ=4.4 nm) and oxygen (543 eV, λ=2.3 nm).  In this energy range, known as the ‘water window,’ photons readily penetrate the aqueous environment while encountering significant absorption from carbon- and nitrogen-containing organic material.  Consequently organic material absorbs approximately an order of magnitude more strongly than water, producing a quantifiable natural contrast image of cellular structures. Since cells are rapidly frozen rather than chemically fixed they are visualized in a close-to-native, fully functional state. We have used SXT to examine to study the effect of antimicrobial agents on the pathogenic yeast, C. albicans (Uchida et al., 2009. PNAS 106:19375-19380) and the cell cycle of diploid and haploid cells of the yeast, S. pombe (Uchida et al., 2011. Yeast 28:227-236), revealing quantitative structural information from statistically significant populations of cells. Most recently we examined the interphase nucleus of primary cells obtained from mouse olfactory epithelium. These studies reveal detailed information about the organization of the interphase nucleus, including the amount and distribution of heterochromatin and euchromatin during interphase. Because the x-ray absorption follows Beer’s Law, the absorption of photons is linear and a function of the biochemical composition at each point in the cell.  Consequently we can accurately measure the degree of chromatin compaction in the heterochromatic and euchromatic regions. To obtain molecular information, we developed a high numerical aperture cryogenic light microscope for correlated imaging. This multi-modal approach allows labeled molecules to be localized in the context of a high-resolution 3-D tomographic reconstruction of the cell.