The Development of a Tabletop Soft X-ray in vitro Microscope

K. Powella, G. Duffya, P. Choia,b, R. Aliaga-Rossela, O. Benalia,b, H. Kelesb, B. Lebertb, O. Sarroukhb, L. Tantarta, C. Zaepffelb, A. Michettec

aNANO-UV sas, 16-18 av du Quebec, SILIC 705, Villebon/Yvette 91140, France bEPPRA sas, 16 av du Quebec, SILIC 706, Villebon/Yvette 91140, France

cDept. of Physics, King’s College London, Strand, London WC2R 2LS, UK Abstract. Soft x-ray microscopy is an attractive tool for the study of biological samples in-vitro, due to the penetrating nature of x-rays and the natural contrast which can be achieved in hydrated samples. There has been a roadblock to the commercialisation and rollout of small, laboratory scale, x-ray microscopes for use in the wider community, as high resolution x-ray microscopy requires tuneable, high brightness x-ray sources. NANO-UV has engaged in a product development programme to introduce the first affordable stand-alone compact soft x-ray microscope for in-vitro studies, known as McXI. The initial specification of McXI is to provide 100 nm resolution on in-vitro specimens, with a unique wavelength selection mechanism in the 2.3-4.4 nm region. Keywords: Soft x-ray, in-vitro, compact, discharge produced plasma, microscope, water window PACS: 68.37.Yz

INTRODUCTION

The advantage of x-ray microscopy in the ‘water window’ spectral region to study complete cellular structure is well known. The major limitation to widespread applications of soft x-ray microscopy (SXM) is the limited availability of sufficiently bright soft x-ray sources that can be integrated into a turnkey instrument.

NANO-UV has developed a unique EUV/soft x-ray source based on a micro-plasma-pulsed (MPP) discharge. The source exhibits radiation brightness comparable to that from a second-generation synchrotron in the region of 100s of electronvolts. This source is being used as the backbone to evaluate the design of a compact standalone SXM product known as McXI (Microscope X In-vitro).

In parallel with this development, a Eurostars Project—McXI E!4885—has been initiated to extend the resolution of McXI down to 20 nm, with improved sample handling and a man-machine interface design to broaden the application range of this standalone microscope. The design and operational data of the source in McXI is presented here.

MCXI DESIGN – OBJECTIVE AND SPECIFICATION

The McXI™ design is a standalone microscope operating in the “water window” soft x-ray region, primarily developed for the study of hydrated samples, such as entire biological cells. The system has sufficient resolution and magnification to allow the study of intracellular features of thick samples, in the microns range, over a sufficiently large field size of 20 m or greater. No specific sample preparation is necessary, and a user friendly specimen sample introduction and manipulation stage has been implemented. The short exposure time that can be achieved, in the range of seconds, avoids mandatory cryogenic sample preparation and manipulation.

The microscope utilizes simple diffractive optics components [1] to provide a resolution of 100 nm. The design permits easy upgrade for resolutions down to 20 nm. A schematic of the architecture of the microscope is shown in Fig.1. Future upgrades provided include options for phase-contrast and other imaging modalities. User-selectable operation wavelength within the 2.3-nm to 4.4-nm region will be implemented to facilitate selective contrast

The 10th International Conference on X-ray MicroscopyAIP Conf. Proc. 1365, 128-131 (2011); doi: 10.1063/1.3625321

© 2011 American Institute of Physics 978-0-7354-0925-5/$30.00

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imaging among organic elements [2]. The microscope includes automated set-up functionality for non-specialists, making it a turnkey industrial product to maximize productivity.

The Eurostars collaborative project McXI E!4885 is a multi-national project supported under the EUREKA umbrella involving NANO-UV (France), Delong Instruments (Czech Republic), and Silson Ltd. (UK). Drawing on the expertise of Delong Instruments in mechatronics and electron microscope design, and the expertise of Silson Ltd. in in vitro sample handling, the aim of the project is to extend the resolution of McXI down to 20 nm, with improved sample handling and man-machine interface design to broaden the application range of this standalone microscope.

FIGURE 1. Schematic of the McXI™ microscope.

CYCLOPS™ – HIGH BRIGHTNESS EUV/SX SOURCE

The CYCLOPS™ is a compact, easy-to-use, reliable, and cost-effective high-brightness SX/EUV light source developed at NANO-UV, based on the patented i-SoCoMo™ technology. The i-SoCoMo™ is composed of a micro-plasma-pulsed (MPP) discharge unit directly coupled to a built-in photon collection and projection unit known as the PlasmaLens™. The source brightness is due to the optical properties of the PlasmaLens™ delivering a narrow beam of clean photons with near zero debris. The high brightness of the CYCLOPS™ provides unique opportunities in the development of EUV and SX metrological instruments.

To quantify the radiation properties downstream from the plasma source, a windowless silicon photodiode with known responsivity (IRD, SXUV 20A) was used with a set of foil filters to create a narrow spectral range centered on the nitrogen He-like line around 2.9 nm. The MPP discharge was operated in an admixture of He and N2 to provide requisite radiation in the ‘water window’ SX band. This is shown in Fig. 2 along with the result of scanning the emitted beam with a small pinhole, placed 62 cm from the source, to obtain a beam-shape profile that has been fitted to a Gaussian distribution.

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(a) (b) (c)

FIGURE 2. The CYCLOPS™ source module performance in the SX region. (a) The spectral region where the transmittance matches the ratio of the single (magenta) and double (green) 526-nm-thick titanium filter on a 75-nm-thick silicon nitride window in the 2.8- to 3.5-nm band. (b) Electrical voltage across the discharge capacitor (top) and SX signal (bottom). (c) Measured beam

profile at 62 cm with Gaussian fit.

SOURCE OPTIMIZATION FOR WATER WINDOW SOFT X-RAYS

Z* is a powerful 2-D magneto hydro-dynamic (MHD) modeling code, developed at EPPRA, to model industrial plasmas with realistic physical boundary conditions. The model features the dynamics of capillary discharge and emission, including electron beam and plasma channeling (ε >> 1); volumetric MHD compression; and where the skin depth >> the plasma diameter and in a highly ionized, fast electron environment. Figure 3 shows the results of the modeling work and how it was used to optimize the source characteristics for soft x-ray emission.

FIGURE 3. Optimizing CYCLOPS™ for soft x-ray radiation. (a) Capillary model. (b) Fast electrons induced discharge in 3-D volumetric compression regime. (c) Time-integrated image of soft x-ray (400 – 600 eV) source. <Z> 5, Te = 45 – 55 eV,

Ne 2·1017cm-3, Nitrogen: He-like and H-like. (d) Current pulse and soft x-ray emission.

RESULTS

To characterize the source for the McXI microscope, measurements were taken for the source size and beam divergence by comparing projected images of test objects located at varied distances from an entrance aperture. The images obtained are shown in Fig. 4 from which the required source parameters were quantified.

R(cm)

Z(c

m)

-0.2 0 0.2

0.5

1

1.5

2

Ne(Av)

3.6E-073.4E-073.3E-073.1E-073.0E-072.8E-072.7E-072.5E-072.3E-072.2E-072.0E-071.9E-071.7E-071.6E-071.4E-071.3E-071.1E-079.4E-087.8E-086.3E-084.7E-083.1E-081.6E-08

t= 1.9037E+01 ns

anode

cathode

cap

illa

ry

cap

illa

ry

Frame 001 13 Aug 2010 ZSTAR - code output, cell values

R(cm)

Z(c

m)

-0.2 0 0.2

0.5

1

1.5

2

Qww(J/ccm)

0.550.520.480.450.420.380.350.320.280.250.220.180.150.120.080.05

time integrated

anode

cathode

cap

illa

ry

cap

illa

ry

Frame 001 13 Aug 2010 ZSTAR - code output, cell values

(a) Simulated capillary

(d) Discharge current and soft x-ray pulse

0.48J/pulse charge

(b) Nitrogen plasma at emission maximum

(c) Time integration of soft x-ray (400 – 600 eV)

Filter Responses1.E-07

1.E-06

1.E-05

1.E-04

1.E-03

1.E-02

1.E-01

1.E+00

1.E+01

1 2 3 4 5 6 7 8 9 10Wavelength (nm)

Log

Tra

nsm

itta

nce

SiN + Ti

SiN +Ti x2

Band Pass

a) 130

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Beam Divergence

0

0.2

0.4

0.6

0.8

1

1.2

0 5 10 15

Axial Displacement (mm)

FW

HM

Beam

Dia

mete

r (m

m)

(a) (b) (c) FIGURE 4. Projection imaging on a CCD detector of an F4 grid test object at different magnifications, through a 10-m aperture located at differing distances, filtered with a 75-nm Si3N4 window. (a) F4 grid at 20.0 mm from aperture, (b) F4

grid at 38.6 mm from aperture, (c) derived plot giving source beam divergence of 0.68°.

CONCLUSIONS

The elements of a tabletop soft x-ray microscope McXI™ developed for the in vitro studies of biological samples have been presented. The microscope is made possible by the unique brightness of the compact CYCLOPS™ source module, which has been optimized for soft x-ray emission and shown to have low beam divergence. Sample manipulation is simplified by the incorporation of a sophisticated sample-handling stage, and elaborate sample preparation is avoided due to the penetrating nature of the x-ray radiation in ‘soft’ materials—unlike electron microscopy—and the short exposure times afforded by the bright x-ray source.

ACKNOWLEDGMENTS

The authors are grateful for the input of our collaborators who include Pontificia Universidad Católica de Chile; RRC Kurchatov Institute, Moscow, Russia; Keldysh Institute of Applied Mathematics RAS, Moscow, Russia; and King’s College London.

We are also grateful for the support of our sponsors, the EU & French Government, Agence Nationale De La Recherche (ANR- EUVIL), OSEO-ANVAR, Eurostars- McXI ! 4885, and RAK Investment Authority (RAKIA).

REFERENCES

1. A. G. Michette, Optical Systems for Soft X-rays, Berlin:Springer-Verlag, 1986. 2. A. G. Michette, A. Erko, M. Idir, and T. Krist, Eds., Modern Developments in X-Ray and Neutron Optics (Springer Series in

Optical Sciences), Berlin:Springer-Verlag, 2008.

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