ABSTRACT
In an ideal world, one would be able to examine the fine-structural details of a cell using a simple, bright-field (BF) light microscope, and cellular imaging would be cheap, fast, and easy to perform. Unfortunately, real-world factors prohibit this Utopian fantasy. The spatial resolution of a light microscope is “diffraction limited”—it is directly proportional to the wavelength of the light (Abbe 1873). For example, using 500 nm green light illumination and an objective lens with a numerical aperture (NA) of one limits the spatial resolution of the microscope to roughly 200 nm, not good enough to visualize detailed subcellular structures (Hell et al. 2004, Huang et al. 2011). In addition, very few cellular structures contain chromophores or fluorophores. As a result, most cell contents are optically transparent towards visible light (Alberts et al. 2008). Of course, specific regions of the cell can be “painted” with dyes, stains, or genetically encoded fluorescent tags and then imaged using, for example, a fluorescence microscope (Chalfie et al. 1994, Tsien 2005, Ellisman et al. 2012, Kilgore et al. 2013). While this approach is enormously informative, and has been responsible for much of our knowledge of cell sub-structure and cellular organization, it is not without perceived obstacles. For example, there is always concern that 818the process of painting can damage or rearrange delicate cellular features, or the paint moiety can bind in the wrong location, or not stick as expected. Any of these can result in images that do not represent the true in vivo organization. Overcoming these shortcomings requires microscopes capable of imaging beyond the diffraction limit of visible light optics and the capacity to visualize unstained biological material. Soft X-ray microscopy overcomes both of these shortcomings of light microscopy (Rudolph et al. 1986, Kirz et al. 1995, Weiß et al. 2000, Larabell and Le Gros 2004). The short wavelength of soft X-rays, compared to visible light, permits imaging at significantly higher spatial resolution. And, as will be described in the following sections, soft X-ray microscopy yields images of cells without the need to paint them with exogenous visualization aids.
