Today’s developments in synchrotron technology continually push the peak brilliance of the X-rays, enabling fast measurements as well as time-resolved in situ experiments. 7 Consequently, radiation-induced sample degradation and the maximum X-ray dose start to become the limiting factors for experiments at these facilities. As described above, one way avoid the dose problem is to scan different spatial position with microfocused beams, or use (microfluidic) continuous flow systems. However, each of these alternatives is limited to certain sample types. 45,142
Another path for overcoming the X-ray dose problems becomes available with the advent of highly intense and ultrashort-pulsed X-ray free electron lasers (XFEL). 6,49 These
free-electron laser sources currently generate X-ray pulses at rates up to 120 Hz (LCLS, SLAC, Stanford, USA) while future facilities will even generate up to ca. 27,000 pulses per second that are bundled in 10Hz bunch trains (European XFEL, Hamburg, Germany, in 2015/2016). These X-ray pulses are so intense that a full diffraction pattern of nanometer-sized crystals is collected within a single shot and that the X-ray beams “are capable of destroying anything in their path”. 6,50,196 During the illumination with a single light pulse, the sample explodes and turns into a glowing plasma (ca. 60,000 K). 196. Due to the ultrashort femtosecond pulse length however, the X-ray pulses outrun the explosion process, ‘freezing’
the atom positions in space. 49,50 An analogue example from the macroscopic world for this
‘freezing’ principle is the highspeed flash photography work done by Harold Edgerton (MIT, Cambridge, USA). For example, his photograph “Cutting the Card Quickly” (1964, 1 µs exposure) shows how a bullet-separated play card levitates in air at its original position.
This concept of ‘diffraction before destruction’ is of great importance for the study of ultrafast processes and the characterization of a wide range of X-ray sensitive samples. 49 As an example, the structure determination of transmembrane proteins is important for medicine and the life sciences because this information can help to understand the signalling of cells or mechanisms of diseases. 6 However, the structure determination of proteins is a very challenging task because these and many other biological samples are only available in small amounts and/or tend not to form single crystals of sufficient dimensions for traditional X-ray (micro-)crystallography. 6 It is much easier to grow nanometer-sized protein crystals, but these cannot be analyzed at tradionional X-ray sources due to their small size and the discussed dose limit. The relation between the average intensity of a diffraction peak and the crystal volume is approximately proportional. 45 Hence, the required beam intensity of a 20 µm crystal is 1000-fold higher than for a 200 µm crystal. 45 Theoretical and experimental results show that the critical dose for a successful structural evaluation is mainly dictated by the crystal size, despite cryogenic attempts of measuring protein crystals that are cooled below 100 K. 45
Recently, femtosecond X-ray protein nanocrystallography has been demonstrated for the high-resolution characterization of photosystem I&II and the model protein lysozyme, showing that the X-ray dose limit can be overcome as described above. 6,197-199 As a consequence of the enormous X-ray pulse intensity, statically mounted samples or experimental environments for flowing samples that are based on closed geometries are incompatible with these 4th generation X-ray sources. Therefore, special sample environments are required that deliver the samples in mid-air and under vacuum conditions while being as sample efficient as possible. 53,200 These kinds of sample environments for XFELs and how microfluidics offers great potential for the adequate delivery of samples will be discussed in this chapter.
Currently, continuously replenishing sample streams are generated using aerodynamic lens particle injectors or liquid jets using a glass-based capillary-in-capillary design. 53,200 Since the
glass capillaries are widely used and suitable for liquid samples, this chapter will focus only on these. The principle for the generation of liquid jet is based on a gas sheath which shapes a liquid stream and has first been demonstrated in a plate-orifice geometry. 201,202 Later, this concept has been transferred to glass capillaries which run essentially clogging free due to their gas-dynamic virtual nozzle design (GDVN). 53 In other words, the pressured gas forms a liquid stream and avoids any wall contact of the liquid. 53 This results in a very stable and reliable system for the generation of nano- or micrometer-sized liquid jets that require only small amounts of sample (down to ca. 100 µl h-1). 203,204
The main drawback of this glass capillary design is the complex fabrication process that involves steps like flame polishing of the tip, its grinding as well as alignment of the inner and outer capillary. 53,203-205 These steps require the manual skills and attention of a lab worker which intrinsically results in geometric variations of the nozzles. It is therefore very hard or impossible to exactly reproduce a targeted design or even automate the process. This is a major issue because the generated fluid dynamics of the liquid jet and, hence, minimum flow rates and liquid jet diameters strongly depend on the geometric and experimental parameters.
205-207
The fabrication procedure is one key point where microfluidic devices shine, due to their fast and easy fabrication and highly reproducible design which is based on established soft-lithographical techniques. 55-57 In this thesis, microfluidic chip-based devices are presented that produce liquid jets with µm-diameters (20 down to 2 µm, or even 940 nm) at very low flow rates (down to 150 µl h-1) under atmospheric or vacuum conditions. These microfluidic liquid jet devices are also based on the gas dynamic virtual nozzle (GDVN) design which enables reliable and essentially clogging-free jetting over long periods of time. 53
The flexibility in microchannel design control is demonstrated by the easy integration of additional microfluidic features, such as jet-in-jet flow focusing, which could enable new in situ experiments at XFELs, or dense arrays of multiple adjacent liquid jet nozzles on a single device, without the need of additional production steps. Hence, the potential of simplifying and up-scaling the fabrication of micro-nozzles for the generation of liquid jets is demonstrated. The microfluidic liquid jet system is highly relevant for the establishment of microfluidics at XFELs because these devices deliver the sample continuously, reliably and efficiently in atmoshperic or under vacuum conditions, as illustrated in Fig.6. 136
Figure 6 Combining the liquid jet principle with microfluidics. (A) Illustration of the building block principle of functional microfluidic tools that can be combined and stacked using the microfluidic liquid jet device principle. (B) The X-ray beam hits the liquid jet in this illustration of the experimental setup. Microfluidic devices are advantageous because each liquid jet device contains a dense array multiple microfluidic GDNV-nozzles which enable fast nozzle changes and, consequently, reduce (expensive) downtimes at the X-ray free electron lasers. (Image adapted and extended from 6)
2.5 Conclusions
In conclusion, the combination of microfluidics with microflocused X-rays is a valuable experimental methodology for the study of fast in situ experiments. While this field is still emerging, a wide range of device types is already available. The on-going development of microfabraction techniques and advent of materials for the production of X-ray compatible microfluidic devices add to the great potential and this technique’s future applications.
Additionally, the variety of other X-ray imaging and spectroscopic techniques could extend the experimental opportunities of microfluidics at X-ray sources even further. Future developments of microfluidic systems and highly brilliant X-ray sources, such as synchrotrons or XFELs, could soon lead to the fundamental understanding of nucleation and growth processes or integration of the high-throughput screening of proteins that yields full three-dimensional as well as dynamics information about these species or even whole cells; with important insights for the natural and life sciences.