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 Technical ReportRAL-TR-98-007Laser Plasma X-Ray Contact Microscopy ofLiving Specimens Using a ChemicallyAmplified Epoxy ResistTMR Large-Scale Facilities Access ProgrammeAC Cefalas et alJanuary 1998COUNCIL FOR THE CENTRAL LABORATORY OF THE RESEARCH COUNCILSLaser Plasma X-Ray Contact Microscopyof Living Specimens Using a ChemicallyAmplified Epoxy Resist AC Cefalas et alNational Hellenic Research Foundation, GreeceAn experiment performed with funding from theTMR Large-Scale Facilities Access ProgrammeContract No. ERBFMGECT950053Laser Plasma X-Ray Contact Microscopy ofLiving Specimens Using aChemically Amplified Epoxy ResistAn experiment performed with funding from theTMR Large-Scale Facilities Access ProgrammeAccess to Lasers at the Central Laser FacilityRutherford Appleton LaboratoryContract No. ERBFMGECT950053AC Cefalas, P Argitis, Z Kollia and E SarantopoulouNational Hellenic Research Foundation, Theoretical and Physical Chemistry Institute, 48 Vas.Constantinou Av.  Athens 11635 Hellas, Greece.TW Ford, AD Stead and A MarrancaRoyal Holloway, University of London, Egham, Surrey TW20 0EX, UK.CN Danson, J Knott and D Neely(G Booth in report preparation)CLF, Rutherford Appleton Laboratory, Chilton, Didcot, Oxon OX11 0QX,  UK.SUMMARYThis report describes the experiment entitled ‘Laser Plasma X-Ray Contact Microscopy ofLiving Specimens Using a Chemically Amplified Epoxy Resist’; carried out at the CentralLaser Facility (CLF) from the 3rd to the 9th February 1997.  The experiment, funded by theFramework IV Large-Scale Facilities Access Scheme, was proposed by Dr AC Cefalas,Theoretical and Physical Chemistry Institute, National Hellenic Research Foundation,Athens, Greece, and carried out by visiting researchers from the Institute.  They weresupported by researchers from the UK from Royal Holloway College, University of Londonand the Central Laser Facility, Rutherford Appleton Laboratory.Experimental Results• Investigated the use of an epoxy novolac chemically amplified photoresist to produce X-ray images of living biological specimens in the water window using laser plasmagenerated soft X-rays (2.4 - 4.4 nm).• The photoresist response was at least one order of magnitude “faster” than the standardPMMA (polymethyl methacrylate) previously used in soft X-ray contact microscopy(SXCM).• Obtained images of biological specimens clearly showed the flagella of the motile greenalga, Chlamydomonas, suggesting a lateral resolution better than 300 nm, whilst the AFMwas capable of discriminating height features of 20 nm in depth profiles.• Perhaps, more significantly however, the experiment has demonstrated that suchphotoresists could be used with less intense X-ray sources and could therefore be the basisof development of a small scale soft X-ray microscope using a small commercial laser.The CLF makes beam time at its facilities available to European Researchers with fundingfrom DG-XII, CEC under the Large-Scale Facilities Access Scheme.  For further informationcontact Dr. Chris Edwards at the CLF.  Tel: (0)1235 445582,  e-mail: c.b.edwards@rl.ac.ukDr Costas Cefalas loads up asample prior to a laser shot.The EU researchers withcollaborators and RALsupport staff.From left to rightTop row: C Beckwith, D Neely,C Cefalas, P ArgitisBottom row: J Knott, A SteadArising Publications“Laser plasma X-ray contact microscopy of living specimens using a chemically amplifiedepoxy resist.”P Argitis, AC Cefalas, Z Kollia, E Sarantopoulou, TW Ford, AD Stead, A Maranka, CNDanson, J Knott, D Neely.Submited to Appl.Phys.Letters.“Single pulse, high resolution X - ray contact microscopy with an advanced epoxy novolacresist.”A Argitis, AC Cefalas, Z Kollia, E Sarantopoulou, TW Ford and AD Stead.SPIE proceedings of the 2nd Gr - I international conference on new laser technologies andapplications, Ancient Olympia, Greece, 1-4 June 1997."Fast, high resolution X - ray contact microscopy with an advanced epoxy novolac resist."AC Cefalas,  P Argitis, Z Kollia, E Sarantopoulou, TW Ford, AD Stead, A Marranca CNDanson, J Knott and D Neely.Central Laser Facility Annual Report, RAL-TR-97-045.“Laser plasma X-ray contact microscopy of living specimens using a chemically amplifiedepoxy resist.”AC Cefalas, P Argitis, Z Kollia, E Sarantopoulou, TW Ford, AD Stead, A Maranka, CNDanson, J Knott, D Neely.Rutherford Appleton Laboratory Technical Report, RAL-TR-98-007"Fast, high resolution X - ray contact microscopy with an advanced epoxy novolac resist."AC Cefalas,  P Argitis, Z Kollia, E Sarantopoulou, TW Ford, AD Stead, A Marranca CNDanson, J Knott and D Neely.Submitted to Euro-CLEO, Glasgow, Scotland, UK, 13-18 Sept 1998.Laser Plasma X-Ray Contact Microscopy of Living SpecimensUsing a Chemically Amplified Epoxy ResistABSTRACTWe report on the use of an epoxy novolac chemically amplified photoresist to produce X-rayimages of living biological specimens in the water window using laser plasma generated softX-rays (2.4 - 4.4 nm). The photoresist response was at least one order of magnitude “faster”than the standard PMMA (polymethyl methacrylate) previously used in soft X-ray contactmicroscopy (SXCM). After chemical development of the exposed resists, atomic forcemicroscopy (AFM) of the relief images obtained of biological specimens clearly showed theflagella of the motile green alga, Chlamydomonas, suggesting a lateral resolution better than300 nm, whilst the AFM was capable of discriminating height features of 20 nm in depthprofiles.INTRODUCTIONSoft X-ray contact microscopy (SXCM) is an interdisciplinary technique that has manyapplications in both life and material sciences. For life sciences SXCM enables theultrastructure of living hydrated specimens to be studied without the need of dehydration orother chemical pretreatment of the living specimen by using suitable pulsed X-ray sourcessuch as laser plasmas1,2). The interest in using soft X-rays, in the so called “water window”(2.3 - 4.4 nm or 280 - 530 eV), is based on the low attenuation at these wavelengths causedby water as compared to the attenuation caused by organic matter. Indeed, just below theoxygen K edge (2.4 nm), 1 m water has only 20% absorption while the carbon containingproteins have distinctively higher absorbance3). Therefore, images with good contrast can beproduced by SXCM, provided that a high contrast photoresist is available. Furthermore, theshorter wavelength of the X-ray light, relative to visible light, allows for better resolution thanwith light microscopy whilst the path length of the 3 - 4.4 nm X-ray photons is 20 timeshigher than that of the 10 KeV electrons. Thus, unlike electron microscopy, images ofspecimens up to 10 µm thick can be obtained if their carbon content is low.To be successful the technique requires the development of sensitive photoresist materials forimage recording; these should have optimised contrast, high resolution and an extendedgreyscale. Up to now the only known photoresist used successfully in SXCM has beenpolymethyl methacrylate (PMMA). This is a high resolution photoresist with good contrastbut it is a relatively slow photoresist and therefore requires a very large fluence of X-rays forimaging. This has limited the range of X-ray sources that can be used, mainly to those oflarge national facilities.Laser plasma sources are devices that can efficiently generate X-rays. The intensity and thespectral distribution of the X-ray emission from the plasma depends on many parameters,such as the energy of the laser pulse, its wavelength, its pulse duration, the focusing of thebeam on the target and the atomic number of the target. In this experiment we used theNd:glass laser facility at Rutherford Appleton Laboratory at 1.06 µm to generate soft X-raysin the water window. Using the same geometry and development procedure for all of ourphotoresist samples, we have compared the performance of PMMA (a positive resist) to twoforms of a novel negative, epoxy novolac photoresist, which we call EPR (Epoxy resist)4).MATERIAL AND METHODSThe experimental apparatus for producing soft X-ray contact images consisted of the lasersource and the vacuum chamber (Target Area 4), where the holder containing the biologicalsamples and the photoresist was placed5), shown in figure 1.  The laser source was theNd:glass laser of the Rutherford Appleton Laboratory that can deliver 16 J at 1.06 µm. Thevacuum chamber was evacuated at a pressure of 10-5 mbar and the laser beam was focusedwith a lens of 40 cm focal length on a planar yttrium foil target (Goodfellow, Cambridge,UK). The X-rays were monitored with a pin photodiode and an X-ray diode array6). Afterinitial tests to compare the sensitivity of PMMA and EPR in the absence of either water or abiological specimen cells of the motile green alga, Chlamydomonas, were placed in a dropletof medium on the silicon nitride window (120 nm thick; FaSTec, Silverstone, UK). Thephotoresist was placed onto this and the holder assembled and tightened to ensure that thespecimens were in close contact with the photoresists1).Fig1. A schematic of the experimental apparatus for producing soft X-ray contact images.                                            X-Rays                                               Biological specimen         waterPhotoresistSi3N4 windowAl foil~100 nmThe thickness of the water was monitored by light microscopy prior to placing the specimenholder into the vacuum chamber. To filter out the 1 KeV photons a thin (0.1 µm) aluminiumfilm was placed in front of the silicon nitride window. Throughout the experiments thedistance between the target and the front surface (i.e. silicon nitride window) was maintainedat 15 mm.A Du Pont Elvacite 2041 (MW 443,000) polymer was used for PMMA resist formulation.500 nm films were spun from a propylene glycol methyl ether acetate solution and prebakedat 160o C for 60 mins. Methyl isobutyl ketone/ Isopropanol 1:1 was used as developer atdeveloping times from 45 secs to 105 secs. The EPR resist formulation described elsewhere4)was used as the standard EPR. The modified epoxy novolac resist version was formulatedwith a higher MW polymer (MW 3300 vs MW 2250). For both the EPR and modified EPRresists, a 4 min post exposure bake at 90o C was used, and methyl isobutyl ketone was used asdeveloper (1 min) followed by a rinse with isopropanol (45 secs). The developed photoresistswere examined with a Burleigh SPM atomic force microscope with a 75 µm scan head.RESULTSInitial images taken either without a silicon nitride window or, when a window was present,without a biological specimen, revealed that the negative, epoxy novolac photoresist, which0.00.20.40.60.81.00 2 4 6 8 10Laser energy (mJ)Normalized thicknessFig.2 Normalised thickness as a function of the input laser energy for the EPRresist.we call EPR4), is at least one order of magnitude more sensitive in getting useful images thanPMMA. A fluence of 50 mJcm-2 in the water window, was found to be the threshold for ablank image to appear on the PMMA, although such images were very shallow, with just 20nm maximum depth difference; this agrees with previously published figures7,8). Incomparison the threshold for EPR in a similar experiment was only 3 - 5 mJcm-2 and fluencessuch as this were obtained with a laser energy of just 300 mJ (Fig. 2). This value was evenlower for a slightly modified epoxy novolac based resist (0.5 mJcm-2) which was producedusing a related material formulated with a higher MW polymer.Images of Chlamydomonas cells were successfully obtained using the negative epoxy novolacphotoresist (Fig. 3), these images clearly show the cell body and the flagella. Using PMMA inconjunction with a similar source to specimen distance, biological imaging was not possiblewhen the specimen was behind an 0.1 µm aluminium filter even at the maximum energypulse obtained from the laser. This suggests that in previous imaging experiments9,10) aFig. 3 Image of Chlamydomonas using the EPR resist. The laser energy was 10.6 J and thedevelopment time was 1 min. The cell body and the flagella are distinct but no structural details arevisible within the cell body.significant contribution to image formation may have been from higher energy photons orfrom UV which is also filtered out by an aluminium coating11).The biological images (Figs. 3,4) recorded with EPR suggest a lateral resolution considerablybetter than 300 nm as the diameter of flagella are approximately 600 nm but these are clearlydistinguishable. For successful biological imaging it is also necessary to be able to distinguishadjacent areas of the specimen which may differ only marginally in their ability to absorb softX-rays, this is in effect the contrast characteristics of the photoresist. The present images,unlike previous studies using PMMA12,13), show no ability to discriminate structures withinthe cell, that is to say the absorption by the cell body was sufficient to result in the completedissolution of the photoresist when it was developed. However, the response of thephotoresist was able to differentiate between a single flagellum and areas where two flagellaoverlapped (Fig. 5). In this case the photoresist area corresponding to the overlapping regionswas c.80 nm deep, whereas images of single flagella were only c.20 nm. Since the depth ofthe images of the cell body were c.100 nm deep it is concluded that two flagella also absorbedsufficient X-rays to produce a maximum response.Fig. 4 Image of Chlamydomonas using the EPR resist. The laser energy was 10.6 J and thedevelopment time was 1 min.Fig. 5 The depth of the area corresponding to single flagella is approximately 20 nm deep(open arrow) whereas those of overlapping flagella are 80 nm.In conclusion we have therefore demonstrated that the novel photoresist used in this study aresignificantly more sensitive than the traditionally used PMMA and, whilst their range ofsensitivity may not be as great as PMMA, such materials can be used in imaging experiments.Perhaps, more significantly however, is that such photoresists could be used with less intenseX-ray sources and could therefore be the basis of development of a small scale soft X-raymicroscope using a small commercial laser.REFERENCES1. T W Ford, A D Stead and R A CottonElectron Microscopy Reviews, 4 269, (1991)2. J Kirz, C Jacobsen and M HowellsQuart. Rev. Biophysics, 28 33, (1995)3. R A Cotton, J H Fletcher, C E Webb, A D Stead and T W Ford SPIE Proceedings, 201586, (1994)4. P Argitis, I Raptis, C J Aidinis, N Glezos, M Baciocchi, J Everett and M HatzakisJournal of Vacuum Science & Technology, B13 (6), 3030, (1995)5. A D Stead, T W Ford, C Danson, D Pepler and M Ebbage Annual Report to the LaserFacility Committee, Rutherford Appleton Laboratory. pp. 47, (1995)6. G Eker, G J Tallents, A Behjat, D Neely, E Wolfrum, A D Stead and T W FordAnnual Report to the Laser Facility Committee, Rutherford Appleton Laboratory. pp. 111,(1996)7. T Tomie, H Shimizu, T Majima, M Yamada, T Kanayama, H Kondo, M Yano and M OnoScience, 252 691, (1991)8. R A Cotton, A D Stead, T W Ford, J H Fletcher and C E Webb SPIE Proceedings, 1741204, (1993)9. T W Ford, R A Cotton, A M Page and A D SteadAnnual Report to the Laser Facility Committee, Rutherford Appleton Laboratory. pp. 52,(1993)10. A D Stead, R A Cotton, A M Page, C G Steele, R Bagby and T W FordAnnual Report to the Laser Facility Committee, Rutherford Appleton Laboratory. pp. 64,(1994)11. T W Ford, R A Cotton, A M Page and A D SteadAnnual Report to the Laser Facility Committee, Rutherford Appleton Laboratory. pp. 52,(1993)12. T W Ford, R A Cotton, A M Page and A D SteadIn: X-Ray Microscopy IV, (Eds. V.V. Aristov & A.I. Erko). Inst. MicroelectronicsTechnology, Chernogolovka, Russia. pp. 276, (1994)13. A D Stead, T W Ford, J A Catcheside, C P B Hills and A RidgeleyJournal of X-Ray Science & Technology 2 172, (1990)

Laser Plasma X-ray Contact Microscopy of Living Specimens using a Chemically Amplified Epoxy Resist

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