5.4 nm spatial resolution in biological photoemission electron microscopy
Introduction
Photoemission electron microscopy (PEEM) combines in a unique way light-optical probing with electron optical image generation. The fundamental process underlying this type of microscopy is the photoelectric effect, which describes the emission of electrons from a solid surface when ultraviolet light is absorbed. PEEM typically utilizes light from synchrotrons, lasers and ultraviolet lamps for specific and gentle probing of physical [1] and biological [2] properties, and for recording fast dynamic processes with temporal resolution down to the sub-femtosecond regime [3]. Many of the experimental methods recently developed for light-optical microscopy in biology can, in principle, also be utilized in PEEM. Indeed, molecular staining and colloidal labeling have already successfully been explored in PEEM [2], [4]. Plasmonic enhancement, multi-photon excitation and emission from nanoparticles bound to biological membranes and proteins are currently being explored. Because PEEM avoids electron beam exposure, damage-free observation in biological applications is less challenging than in conventional electron microscopy. In most applications the photoelectrons are emitted with low efficiency and with small kinetic energies. To obtain bright images and high resolution, acceleration to energies of the order of a few 10 kV is a necessity. This acceleration produces strong optical aberrations which, in combination with lens aberrations, have up till now limited the resolution in PEEM to approximately 8 nm. While this resolution is better than that of all other light-optical techniques, including super-resolution techniques [5], [6], it could further be improved with an aberration corrector. With such corrector brightness, contrast and sensitivity would be enhanced as well. There are, however, only few practical ways to correct the aberrations occurring in electron optical systems [7]. Scanning and transmission microscopes utilize multi-pole lenses as electron optical correctors [8], [9]; for PEEM mirror-based correctors are considered more suitable and have recently been built at Berkeley [10], BESSY-Berlin [11], and in Portland [12]. In this paper we report a first significant step in improving the resolution of aberration-corrected PEEM. Utilizing a hyperbolic electron mirror we demonstrate a resolution of ∼5 nm for biological specimens. Our experimental and theoretical results indicate that the developed corrector may ultimately bring the resolution to below 2 nm, which would close the gap to today’s scanning electron microscopes.
Section snippets
Results and discussion
Fig. 1 shows the overall lay-out of the microscope and the details of the correcting mirror. The mirror is operated in a symmetric mode, in which the outgoing electrons follow the same trajectories as the incident electrons, only with reversed direction. In operation the mirror produces negative spherical and positive chromatic aberration coefficients; these are just opposite in sign to those occurring in the lens system and in the acceleration process near the sample surface. Quantitative
Conclusions
Using an aberration-corrected photoemission electron microscope we have achieved 5.4 nm resolution in the imaging of biological samples. To our knowledge this is the best resolution reported for PEEM to date and for any microscopic techniques using light-excitation in biology. This high-resolution work used an ultraviolet laser for illumination which caused no discernible structural damage in 30 min of experimentation. In-situ experiments and numerical results indicate that an ultimate resolution
Acknowledgments
Technical support by W. Skoczylas, P. Witham, T. Dornan and M. Nisenfeld at PSU is gratefully acknowledged. This research was supported by NSF under Grant number DBI-0352224 and by DOE under DE-FG02-07ER46406.
References (14)
Image properties in an aberration-corrected photoemission electron microscope
Phys. Procedia
(2008)- et al.
Photoemission electron microscopy
- et al.
Photoelectron microscopy and immunofluorescence microscopy of cytoskeletal elements in the same cells
Proc. Natl. Acad. Sci. USA
(1983) - et al.
Attosecond nanoplasmonic-field microscope
Nat. Photon.
(2007) - et al.
Silver-enhanced colloidal gold as a cell surface marker for photoelectron microscopy
J. Histochem. Cytochem.
(1986) Super-resolution microscopy: breaking the limits
Nat. Methods
(2009)- et al.
High-resolution near-field Raman microscopy of single-walled carbon nanotubes
Phys. Rev. Lett.
(2003)