Modifications for soft x-ray contact microscopy: quantification of carbon density discrimination and stereo imaging

Soft x-ray contact microscopy successfully images hydrated biological material with resolution superior to light microscopy. Nanosecond laser pulses record the image before movement or radiation damage can occur, eliminating concerns of fixation-induced artifacts (cf electron microscopy). X-rays make the recording polymethyl methacrylate (PMMA) photoresist more soluble, thus a contour map of x-ray absorbency is produced in which relative heights, measured by atomic force microscopy, reflect specimen carbon density. Until now quantification of the carbon-density differences was impossible, neither has the minimum carbon density difference which is detectable been determined. Since biological specimens are composed of structures differing only marginally in carbon density the discrimination between carbon densitites is critical. Using SI 3 N 4 windows coated with differing carbon thicknesses we have followed the rate of PMMA dissolution in order to produce calibration curves from which specimen carbon density can be determined. These experiments have also attempted to determine the minimum detectable carbon density difference. When using relatively thick (< 5micrometers ) specimens image interpretation can be difficult as spatially separated structures in the original specimen become superimposed in the x-ray image. To provide spatial resolution in three dimensions we are developing a soft x-ray stereo imaging system. Using two laser plasma x-ray sources and contoured photoresists we have obtained two simulanteous images of the same specimen from different angles. E-beam lithography, cutting and imprinting have been tested as means of producing contoured photoresists. The merits of each will be discussed and preliminary stereo images of hydrated biological specimens presented.