High resolution live cell imaging with electron-beam assisted(EXA) optical microscopy
Yoshimasa Kawata1,3, Yasunori Nawa1, and Wataru Inami2,3
12

Shizuoka University, Faculty of Engineering, Johoku, Naka, Hamamatsu 432-8561

Shizuoka University, Global Research Leaders, Johoku, Naka, Hamamatsu 432-8561
3JST, CREST
Abstract

We propose electron beam excitation assisted optical microscope, and demonstrated
its resolution higher than 50 nm. In the microscope, a light source in a few nanometers
size is excited by focused electron beam in a luminescent film. The microscope makes it
possible to observe dynamic behavior of living biological specimens in various
surroundings, such as air or liquids. Scan speed of the nanometric light source is fasterthan that in conventional near-field scanning optical microscopes. The microscope
enables to observe optical constants such as absorption, refractive index, polarization,and their dynamic behavior on a nanometric scale. The microscope opens new
microscopy applications in nano-technology and nano-science.
In conventional near-field optical scanning microscopes [1, 2] (NSOMs) capable ofsub-diffraction limit resolution, confinement of optical illumination area on thespecimen is realized by small aperture of metal-coated fiber probe, or aperture-less tip
probe, optically-trapped gold particle, solid immersion lens, non-optically probing
technique. Images are acquired by scanning the small aperture near specimen surface
while keeping a constant distance from the specimen surface.
Here, we propose electron beam excitation assisted optical (EXA) microscope inwhich electron beam focused on a luminescent thin film excites nanometric light sourcenear the specimen [3, 4]. The light emission is well known as cathodoluminescence. It is
possible to excite a light source of a nanometric size, because electron beam can befocused to a few nanometers. EXA microscope combines scanning electron microscopythat has nanometric resolution and optical microscopy that is advantageous to
dynamic observation of living biological specimens.
Figure 1(A) shows schematic diagram of the proposed EXA microscope. An
electron beam is focused on a luminescent film. A specimen is put on the luminescentfilm directly. The inset in Fig. 1(A) shows magnified image of the luminescent film andthe specimen. Nanometric light source is excited in the luminescent film by the focusedelectron beam. The nanometric light source illuminates the specimen, and the scatteredor transmitted radiation is detected with a photomultiplier tube (PMT). The light sourceis scanned by scanning of the focused electron beam in order to construct on image.
Figure 1(B) shows the structure of sample holder used in EXA microscope. A square
aperture of 100 × 100 μm2 size was fabricated on silicon substrate and sealed by SiN
film with a thickness of 50 nm.

Fig. 1. (A) Schematic diagram of EXA microscope (A) and layout of silicon nitride
membrane (B).
To verify feasibility of the high resolution EXA microscope, we used silicon nitride(SiN) film of 50 nm thickness as a luminescent material and all optical components
were inserted in vacuum chamber of scanning electron microscope (SEM) (JEOL,JSM-6390). SiN film emits blue to ultra-violet (UV) light by irradiation of electron
beam. We confirmed in a preliminary experiment that SiN film of 50 nm thickness hadenough strength to separate 1 atm air pressure from vacuum.
In the experiment, electrons were accelerated with 10 kV voltage and focused to a
8nm diameter spot on the SiN film to excite nanometric light source. Scattered ortransmitted light from the specimen was collected by a lens with numerical aperture
0.65 and detected with a photomultiplier tube (PMT) (Hamamatsu Photonics,
R7400-U20). Images were reconstructed from the signal detected with raster scanningof electron beam using a computer. We used polystyrene latex spheres dispersed on theSiN film directly as specimens for resolution veri- fication. The latex spheres weredispersed in monolayer, which was confirmed with an atomic force microscope (Seiko,SPI-3800).
Figure 2(A) and 2(B) show observation images acquired with the SEM and EXA
microscopes, respectively. The two images were acquired successively. The scale bars
in the images represent 100 nm length. The acquisition time of EXA image was 16
minutes at 512 by 512 pixels. The resolution and signal-to-noise ratio of the SEM imagein Fig 2A is poor because the SEM image was acquired through the SiN membrane.
Each latex sphere of 100 nm diameter was observed clearly and its position in the EXA
image was identified with that in the SEM image. This allows us to conclude that EXA
microscope has resolution higher than the diffraction limit [23].

Fig. 2. (A) EXA microscope image of 100 nm latex spheres and (B) SEM image ofthe same area as in (A).
Figure 3(A) shows observation results of latex spheres of 50 nm diameter with
EXA micro- scope. Figure 3(B) shows intensity distribution on the solid line indicatedin Fig. 3(A). It is recognized that EXA microscope can resolve aligned spheres of 50 nm
diameter clearly. We believe that the developed EXA microscope has a potential toachieve a few tens nanometer resolution because electron beam can be focused in a fewnanometers.

Fig. 3. (A) Observation image of 50nm diameter latex spheres with EXA
microscope and (B) intensity distribution on the solid line indicated in (A).
We proposed a new type near-field optical microscope and demonstrated its
resolution higher than 50 nm. The concept of EXA microscope is an integration of ahigh-resolution SEM and an optical microscopy suitable for observation of living
biological specimens and spectroscopic analysis of materials. Due to the conversion of
the electron beam to light source, specimens can be placed in air or liquids, as well asvacuum. Since electron beam can be scanned at a fast speed, it is possible to observe
dynamic behavior of specimens with fast frame rate. We expect that the EXA
microscope can resolve the scanning speed limitation of conventional NSOMs due tolow signal-to-noise ratio at higher resolution, and difficulty of maintaining the probespecimen distance.
[1] D. Pohl, W. Denk, and M. Lanz, “Optical stethoscopy: Image recording withresolution λ /20,” Appl. Phys. Lett. 44, 651–653 (1984).
[2] E. Betzig and M. Isaacson, “Collection mode near-field scanning opticalmicroscopy,” Appl. Phys. Lett. 51,2088-2090 (1987).

[3] W. Inami, K. Nakajima, A. Miyakawa, and Y. Kawata, “Electron beam excitation
assisted optical microscope with ultra-high resolution,” Opt. Exp. 18, 12897-12902(2010).
[4] Y. Nawa, W. Inami, A. Chiba, A. Ono, A. Miyakawa, Y. Kawata, S. Lin, and S.
Terakawa, “Dynamic and high-resolution live cell imaging by direct electron beam
excitation,” Opt. Exp. 20, 5629-5635 (2012).