Laboratory Sources for Soft-X-Ray Imaging of Hydrated Biological Material

Abstract

Soft x-rays have been shown to be useful for imaging thick biological sections or even whole cells at better than 50 nm resolution with useful native contrast and without the need for staining. 1 Soft x-ray radiation within the so-called "water-window" energy regime (between the absorption edge of C (285 eV) and O (540 eV)) produces strong contrast from intercellular components but is ~10X less attenuated by surrounding water. 2 The brightest and most intense sources for soft-x-ray radiation are by far synchrotrons which can produce sub-second images at wavelengths tunable through the soft-x-ray regime. In comparison, the few laboratory sources available currently are typically limited to one or a few wavelengths and exposure times are source-brightness limited to many seconds or minutes. 3 A laboratory-based (aka "compact") soft-x-ray microscope with sufficient imaging performance has distinct advantages in cost, size, and ability to be located close to the biological laboratory. This paper compares the laboratory-based soft-x-ray sources we have investigated for both 2D and 3D imaging of hydrated biological material in the 27-100 nm resolution range.The most straightforward method of soft-x-ray generation is ionization matter by a beam of electrons. We have used an SEM probe at 5 keV to create a focused spot of 50-100 nm on a target to produce a soft x-ray point source (Fig. 1). Using probe energies below 5 keV effectively shut off the V/Ti/Sc Kα lines leaving a sharp Lα line (V: 511 eV, Ti: 452 eV, Sc: 395 eV) in the soft-x-ray range. The spot is then used as a pointprojection x-ray source with magnifications of 10-1000+X on to a soft-x-ray sensitive CCD detector ((20µm) 2 1.7Mpixel) 100-300 mm away. An advantage of this technique is the lack of x-ray optics which are costly, complex, delicate, and inefficient. As a result, living cells imaged with the lens-less technique can survive 10-50X longer compared to systems using Fresnel zone plate (FZP) optics. Other aspects of the point projection method include essentially infinite depth-of-field, an inherent phase contrast condition (that must be phase retrieved at these energies to preserve resolution), and cone-beam (half-angle 50 mrad typical) imaging parameters. 4 A 1µA beam current produces reasonable images (2X binning) of dehydrated samples in ~1 minute with a resolution of ~100 nm. Images with 300 photons/pixel through 5µm and 10µm of vitreous ice are expected to take ~5 min and ~20 min when the system is fully functional in spring 2008. A resolution of ~70 nm should be achievable using the V target with a 5X increase in imaging time (although demonstration pending). Ultimate resolution from Ti is expected to be ~80nm and Sc to be ~100nm due to lower density.Laser-pulsed plasma (LPP) sources permit significantly greater source brightness, deliver higher radiance to the sample and permit shorter exposure times. 5,6 In our second generation construction (full functionality expected summer 2008) an ultrafast laser (1064 nm, 800 ps, 180 mJ, 300 Hz) is focused...