The Development of an Automated Nano Sampling Handling System for Nanometre Protein Crystallography Experiments P. Docker+ , D. Axford+, M. Prince*, B. Cordovez~, J. Kay+, D. Stuart+, G. Evans+ + Diamond light Source Harwell OX110DE *Aston University Birmingham B4 7ET ~ Optek Systems Philadelphia, PA, 19104 ABSTRACT As the world’s synchrotrons and X-FELs endeavour to meet the need to analyse ever-smaller protein crystals, there grows a requirement for a new technique to present nano- dimensional samples to the beam for X-ray diffraction experiments.The work presented here details developmental work to reconfigure the nano tweezer technology developed by Optofluidics (PA, USA) for the trapping of nano dimensional protein crystals for X-ray crystallography experiments. The system in its standard configuration is used to trap nano particles for optical microscopy. It uses silicon nitride laser waveguides that bridge a micro fluidic channel. These waveguides contain 180 nm apertures of enabling the system to use biologically compatible 1.6 micron wavelength laser light to trap nano dimensional biological samples. Using conventional laser tweezers, the wavelength required to trap such nano dimensional samples would destroy them. The system in its optical configuration has trapped protein molecules as small as 10 nanometres. Keywords: microfluidics, protein crystallography, X rays, optical nano traps. 1 INTRODUCTION Typically, workers in the synchrotron community look to employ a macro, top-down approach using robots to automate human protocols [1]. This is a methodology often seen in the micro and nano engineering community at large. Currently, as the handling of nano crystals is beyond top down capabilities, X-FEL facilities are currently employing an injector approach. This requires micro litre quantities of reagant containing nanocrystals to be injected in front of the X-ray beam and the diffraction pattern is imaged by the detector. Such systems have a reported ‘hit’ rate of less that 10 % [2]. This paper details optical nano tweezers which are very much a bottom-up approach that facilitates self-assembly, alignment and subsequently automation. This also offers the potential to inform the user that a crystal has been trapped prior to interrogation. This techneque will also present a static array of crystals to be interrogated by the X- ray beam. This technique is not to be confused with traditional optical tweezers which can be used to manipulate a single sample. Although a very useful technique it can only be used with >1 µm sized samples as the wavelength of laser light used to manipulate the sample must be of the same order of magnitude. To achieve the trapping of nano crystals using this technique would require a wavelength of laser light with too much energy thereby destroying any sample it traps. These nanotweezers have been developed by Optofluidics (PA, USA). By using optical wave guides 1064 nm biologically friendly laser light can be used to trap samples sizes down to 10 nm at predetermined sites micro machined into the waveguides. These can be prealigned to the X-ray source and no further alignment would be required. 2 THE TECHNOLOGY This potential solution is offered by Optofluidics, based in the Philadelphia, USA. They have developed technology which employs a microfluidic cell that uses silicon nitride waveguides to overcome the free space limitations of traditional optical traps to capture sub-micron particles with laser light using a wavelength that is compatible with biological samples (1064 nm) whilst maintaining protein sample integrity. Briefly, light is tightly confined at the near-surface of the silicon nitride waveguides, and the technology uses the evanescent wave (light outside of the waveguides) to generate strong optical gradients which are necessary to capture such small particles. If traditional optical trapping technology were to be implemented, such technologies would either be unable to optically capture the nano crystals (because of the light diffraction limit limitations of free space traps) or they would introduce a significant heating effect tothe surrounding volume, likely destroying the analyte. The flow cell allows accurate fluidic delivery (bringing the particles to the trapping locations via pressure driven flow), and once the particles are in the vicinity of the silicon nitride traps, the light is guided through the traps which results in the particles being captured (figure 1).