Abstract

Soft X-ray contact microscopy (SXCM), has many applications in both life and material sciences. In the case of life sciences SXCM enables the study of the ultrastructure of living hydrated specimens, without the need of dehydration or other chemical pretreatment, by using suitable pulsed X-ray sources such as laser plasmas [1,2]. The interest in using soft X-rays, in the so called “water window” (2.3-4.4nm), is based on the low attenuation at these wavelengths caused by water, as compared to the attenuation caused by organic matter. Therefore, good contrast masking of the incident radiation is provided. The successful imaging of biological specimen, requires the development of sensitive photoresist materials for image recording; these should have capabilities of high resolution lithography and an extended grayscale. Up to now, the only known photoresist used successfully in SXCM has been polymethyl methacrylate (PMMA). This is a high resolution photoresist when exposed to e-beam or X-ray radiation, with contrast suitable for gray scale recording ; nevertheless, it is a relatively slow photoresist and, therefore, requires a very large fluence of X-rays for imaging. The work reported here was carried out using the Vulcan Nd:glass laser facility at the Rutherford Appleton Laboratory UK, whose rod chain output can deliver more than 11 J at 1064nm. This can be delivered on a Yttrium target as an X- ray source. A very sensitive e -- beam photoresist, used for the first time in SXCM, enabled the biological imaging with the specific source in single pulse experiments in the water window spectral range. This photoresist is an epoxy novolac based chemically amplified photoresist (EPR) which has been proven capable of resolving sub tenth micron features. Initial experiments to compare the sensitivity of PMMA and EPR were done in the absence of a biological specimen. The first image for EPR is obtained at 300mJ laser pulse energy. PMMA used as a reference gave a first image of 40nm depth difference between exposed and unexposed areas, as measured with a Dektak profilometer, at 4.6 J laser pulse energy and an image of 70 nm at 10.6 J. Thus on the basis of the calculations of X-ray flux produced at different laser energies, the minimum flux for image production with PMMA is 2.5mJ.cm^-2 and the corresponding value for EPR is only ~ 0.07 mJ.cm^-2, giving a difference of two orders of magnitude approximately, for the two materials. In biological imaging experiments the living specimens were cells of the motile green alga, Chlamydomonas, which were placed in a droplet of medium. In the experiments with biological specimens no image was obtained with PMMA as a recording material, even with the higher pulse energy available and careful adjustment of water layer thickness in order to be exactly the size of cell diameter. In the contrary, with the EPR resist biological imaging was possible. Images of Chlamydomonas cells were successfully obtained. These images clearly show the cell body and the flagella, suggest a lateral resolution considerably better than 300nm.

© 1998 IEEE