Nanostructured imaging of biological specimens in vivo with laser plasma X-ray contact microscopy

Soft X-ray contact microscopy (SXCM) enables the study of the ultrastructure of living hydrated specimens, without the need of dehydration or any other chemical pretreatment, by using suitable pulsed X-ray sources such as laser plasmas. 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 grey scale. A very sensitive 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 subtenth-micron features. The photoresist response was at least two orders of magnitude 'faster' than polymethyl methacrylate (PMMA), which is the standard resist used so far in SXCM. Atomic force microscopy (AFM) and scanning electron microscopy of the biological specimen images recorded in the resist clearly showed the flagella of the motile green alga, suggesting a lateral resolution better than 100 nm. The resist was also capable of providing height features, as small as 20 nm, in AFM depth profiles and discriminating the flagella intersection areas