Determination of the radiation barrier

The ultimate resolution of X-ray diffraction microscopy and transmission electron microscopy in structural studies of biological specimens is limited by radiation damage. An approach to overcome the degrading effects of radiation damage would be to record diffraction patterns or images in a shorter time than the time of the damage process itself. The detailed molecular dynamics analysis of Neutze et al. [1] gave the first insight that atomic resolution might be possible if femtosecond pulses (in this case from an X-ray free electron laser) were used. Experiments validating the theoretical considerations or determining the ultimate radiation barrier haven’t been done so far. However, for every subsequent experiment with ultrafast electron pulses, e. g. single molecule imaging, it would be imperative to determine the onset of radiation damage precisely. It is furthermore unknown if the total amount of electron dose cn be increased by incresing the time intervals, maybe essential for a recovery mechanism (recombination of broken bonds) of the structure, between the exposures (respectively pulses). With the possibility of using pulses in the femtosecond regime this effect could be studied in detail. This way the prospects of entering the realm of time-resolved structural studies could be investigated and maybe verified.

The future vision of femtosecond pulses used for diffraction or even imaging studies of macromolecular assemblies of single molecules, where an intermediate sized transmission electron microscope might be used, pulsed by a femtosecond source [2,3], are based on this initial study of defining the radiation barrier. If we can overcome the almost intrinsic limiting factor of radiation damage in structural biology, the door to experiments in all 4 dimensions will be opened: deciphering not only the static but also the dynamic strcture of biological substances.

For this first project period we propose to expose biological structures, such as micro- or nanocrystals made from proteins or polymers with femtosecond electron pulses and to study the amount of structural alteration with the help of mid-infrared microspectroscopy. The high spatial resolution will enable us to investigate the alterations of the secondary structures dependent on the pulse length and the pulse frequency.

The mid-infrared spectra of exposed biological samples need to be measureed in situ. Therefore a mid-infrared transmission spectrometer with microscopic focusing and scan stage will be implemented within the environment of a femtosecond electron source operated under high-vacuum conditions, to come possibly near the conditions of the ultimate goal of a strctural analysis of single molecules with femtosecond pulses. The high-brightness mid-infrared beam covering at least the amide vibrational spectrum of protein (5.8 – 7 μm) will be obtained by difference-frequency generation of the output beam of a broadband fs laser operating near 2 μm wavelength, as proposed in project C.3.5  and  A.2.1.