Coherent and incoherent imaging of biological specimens with electrons and X-rays

Highlights

• Damage is proportional to fluence and limits the achievable spatial resolution for X-rays and electrons.

• To distinguish biological materials in a prokaryotic cell absorption contrast is better than coherent elastic scattering for soft X-rays in the water window.

• For higher energy hard X-rays coherent elastic scattering provides the best contrast.

• For energies 20 keV and above the limitation is source brightness not damage.

• Electrons interact more strongly and interpretable phase contrast is limited by the applicability of the weak phase object approximation.

• Phase randomization from multiple scattering means that amplitude contrast dominates for electron scattering from biological objects larger than about 100 nm.

• Differential absorption at Na, Ca and K edges can only be used to determine local concentrations in the molar range, relevant for biomineralization, even for voxels up to 100 nm across.

Abstract

Since radiation damage is proportional to fluence, radiation damage limits the spatial resolution of biological structures determined by either X-ray or electron scattering. If only elastic scattering is used for structural information then electrons are superior as the ratio of elastic to inelastic scattering is higher than for X-rays. For soft X-rays in the water window below the O K edge photoabsorption contrast might be better than elastic scattering for distinguishing different biological materials. Phase contrast elastic scattering is most effective in the hard X-ray region up to about 10 keV. Radiation damage limits spatial resolution for most X-ray imaging to 10-20 nm. Local molar concentrations of Na,K and Ca ions can be determined at somewhat lower spatial resolutions using relevant absorption edges. At higher energies resolution res is only limited by the fluence available from the light source, since energy deposition is small.

Introduction

Imaging using ionizing radiation, whether it is electrons or X-rays, inevitably results in damage that destroys the object under investigation. This problem is particularly acute for biological specimens that are known to be radiation sensitive. The aim should therefore be to maximize the scattering from processes that give structural information, as opposed to the scattering processes that lead to damage and structural disintegration. This is also raises the question on what type of radiation gives the most information with the least damage. This was addressed in a classic paper by Henderson [1] on what radiation is best for near atomic resolution of biological structures. He concluded that electrons were superior to X-rays, since the ratio of elastic scattering that conveys structural information to the energy deposited that leads to damage is much higher for electrons. The inherent flexibility of the electron microscope and the coherence of electron sources meant that it is possible to use either phase contrast or amplitude contrast. Conventional single particle cryo electron microscopy, for which the 2018 Nobel was awarded to Dubochet Frank and Henderson, explicitly uses phase contrast. In an earlier work I compared conventional phase contrast with annular dark field amplitude contrast for biological specimens [2]. I concluded that phase contrast was limited to objects smaller than about 30 nm, due to the breakdown of the weak phase object approximation used to derive structural information, and that for larger objects annular dark field was superior. Hohmann-Marriott [3] and colleagues had already demonstrated the annular dark field could be used to localize heavy element labels in plastic sections. Wolf et al [4] showed that cryo scanning transmission electron microscopy (STEM) was superior for tomographic imaging of complete cells. This led to the theoretical exploration of contrast and how to optimize the range of collection angles by carefully choosing the inner cut off angle for different thicknesses [5].

Traditionally X-ray imaging has only made use of incoherent imaging due to the difficulties of focusing X-rays. A zone plate is used to from a focused probe analogous to the probe in a STEM and this is scanned over the surface of the specimen while the change in the transmitted or some secondary signal is monitored. This is done by moving the specimen stage rather than shifting the beam, as is the practice in the electron microscope. It is particularly valuable for detecting the presence of an element due to changes in the absorbance just after an absorption edge. An alternative method to produce an energy-selected image is to use the zone plate to focus X-rays after they have been transmitted through the specimen, analogous to energy filtered TEM.

Synchrotrons with limiting apertures made it possible to use X-rays for coherent as well as incoherent imaging [6]. Recently coherent imaging has been given considerable impetus by the development of the X-ray free electron laser (XFEL). Although traditionally XFELS are exotic instruments at large synchrotrons facilities such as Stanford or Berlin; a new compact XFEL is under development at ASU that will make coherent X-ray imaging more widely available. XFELS produce short pulses of X-rays that make it possible to outrun radiation damage by acquiring the diffraction data before radiation damage takes place [7]. The availability of synchrotron sources has led to a number of theoretical studies on X-ray imaging, mainly concerned with the resolution achievable at a given dose. In diffractive imaging the object is reconstructed from diffracted intensities in an iterative procedure using a support constraint. Howells et al [8], Shen et al [9] and Starodub et al [10] considered the effects for diffractive imaging of a coherently illuminated macroscopic object. Of particular interest was how the radiation dose varied with the achievable spatial resolution. This was addressed by a number of authors [8,[11], [12], [13], [14]]. Du and Jacobsen [11] also considered both coherent and incoherent imaging and made some comparisons with electrons, and Villanueva-Perez et al [15] investigated incoherent imaging by Compton scattering which is relevant for higher energy X-rays.

Cell components can loosely be classified as water, protein, genetic material, lipids and phospholipids and sugars. There is also the issue of the size of the cell and penetration by X-rays of different energies. Prokaryotic cells from bacteria are about one micron in size; eukaryotic cells from more complex organisms can be ten microns or more in size. It is therefore appropriate to now examine what advantages coherent X-ray imaging might have over incoherent imaging to distinguish these different components and to unify the treatment of both X-ray imaging and electron imaging. Ions such as Na, K and Ca play an essential role in metabolic processes and it is also of interest to determine what local concentrations might be detectable.