A.1-Electron Microscopy


Electron microscopy (EM) covers the "imaging gap" (Fig.1) between high-resolution structural techniques (X-ray, crystallography) and light microscopy, which makes it indispensable to understand the ultrastructure of cells and tissues. Moreover, EM is the highest resolution imaging technology available capable of obtaining unique structures of individual cells, organelles, flexible macromolecules and certain viruses. There are many different sample preparation techniques and imaging modalities within the electron microscopy realm depending on the biological questions at hand. Producing a survey of all of them is beyond the scope of this presentation and the research goals described in this webpage (check the wikipedia entry for a good summary).

Figure 1: Schematic of electron tomography principle. (Left) 2D images are acquired from different angles by rotating the speciment in the sample holder along a tilt-axis. (Right) According to the Projection-Slice Fourier Theorem each 2D projection represents a plane (or slice) in the 3D Fourier space representing the object. Thus, since each projection comes froma  different angle we can recover the 3D Fourier representation of the object and transform into a density map. [Figure from McIntosh, et al. (2005) Trends Cell Biology 15:43-51]


My current projects focus on electron tomography (ET) (Fig. 1), with a strong emphasis in cryo-ET (Fig. 2). Briefly, ET is comparable to medical tomographic techniques like CAT, PET and MRI in the sense that it provides a 3D view of an object, yet it does so at a cellular scale and with nanometer resolution. In particular, CET is capable of obtaining 3D reconstructions at “molecular resolution” (approx. 4-8nm) in situ of intact cells, since cryo-samples are not chemically treated or physically deformed during the imaging experiment. However, quantitative image analysis of CET datasets is extremely challenging due to very low signal to noise ratio (well below 0dB), missing data and heterogeneity of biological structures.

Image courtesy of Luis R. ComolliImage courtesy of Luis R. Comolli

Figure 2: (Left) 2D cryo-electron microscopy image of a mutant of bacterium Caulobacter crescentus. (Right) 3D rendering from cryo-electron tomogram after segmenting of inner membrane (yellow), outer membrane (orange) and cytoplasm (red) . Images courtesy of Luis R. Comolli.