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- Infrared microspectroscopy
- Far-infrared and high resolution FTIR
- Macromolecular crystallography (protein crystallography)
- Powder diffraction
- SAXS / WAXS
- Soft x-ray spectroscopy
- X-ray absorption spectroscopy
- X-ray fluorescence microprobe (x-ray microspectroscopy)
- Beamline fact sheets
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| Samples for SAXS / WAXS |
Samples for SAXS / WAXSThe SAXS/WAXS beamline can provide information on the structure of samples on length scales from approximately 0.1 to 500 nm. SAXS/WAXS is a powerful technique for nano-scale analysis of solid and liquid samples in an enormous range of research fields. For examples of the research conducted at the beamline perusing the Publications web page is recommended. In particular, the beamline facilitates SAXS/WAXS experiments that are not feasible using laboratory equipment. These include time-resolved, spatially-resolved and anomalous scattering (energy-resolved) analysis, grazing incidence scattering, very small specimens, and weakly scattering systems. With its very high incident intensity, specially-designed optical system and endstation capabilities, the beamline is particularly suitable for in-situ experiments. Previous experiments have included observation of changing samples at over 10 frames per second and protein solutions of concentrations as low as 0.1 mg/mL. If you are unsure of the suitability of your sample, please contact beamline staff. When considering an experiment, please be mindful of sample mounting and handling capabilities and of the sample environment required. Proposals for beamtime are required to specify details of sample mounting and environment in the experimental section. The requested sample environment and details of any additional equipment must also be stated in the Detailed Experiment Plan form submitted prior to an experiment. Beamline staff will attempt to accommodate all reasonable requests with regard to providing sample mounts and sample environments for the beamline. Many user-provided systems can be accommodated and the more communication with staff the better we can assist. The available facilities are detailed below under Sample Mounting and Sample Environments. If you require further information, have any questions or would like to discuss a specific sample requirement, please contact beamline staff. Ensuring that you are able to run the samples in a timely and appropriate manner is key to a successful experiment and we request that you give this due consideration when preparing for an experiment. Index to sections below
Sample MountingSample EnvironmentStages - Rotation, Linear, Syringe Pumps Sample Mounting
Sample alignment is performed using areaVision software (developed in-house). An on-axis sample viewing camera provides a direct, live view of the sample before, during and after exposure. Initially the beam position is established by viewing a scintillating crystal placed at the sample position. The beam position is marked with a cursor on the sample view and samples may then be aligned with the beam using the now fixed beam cursor as a reference. The camera can readily view samples of 10 micron size or greater. Time lost due to alignment is minimised because the continuous, live view means no adjustment is required between aligning and exposing samples. The sample stage has a precision of greater than 5 microns in all 3 axes, allowing careful sample alignment. Rapid, precise alignment is further facilitated by the point & click function of the areaVision software, which allows a desired sample position to be aligned with the beam cursor with a single mouse click. Additional, broader views of the sample position, sample mounts or samples themselves can be readily provided with Flea video cameras mounted and positioned as required. These cameras are typically used to provide a view of which sample of a series is positioned in the beam, read sample labels or monitor equipment inside the beamline hutch. A Sony SNC-RZ30P camera is mounted in the beamline hutch to provide remote monitoring of the beamline, and may also be used to view samples and equipment. Back to Index. Solid SamplesDiscrete solid samples, i.e. not powders, may be mounted on a sample plate, similar to that pictured below, that has a grid of holes across which samples are mounted. This reduces the time lost due to sample changes. A range of different plates featuring different hole patterns are available: 1cm diameter holes are available in patterns of 12 x 11 (pictured below), 7 x 8 and 8 x 8 grids while 2cm diameter holes are available in a 4 x 9 grid. Multiples of each type of plate are available, allowing a series of samples to be mounted while data are collected on another. Mounting samples on plates the day before an experiment is recommended where possible to allow efficient use of beam time.
Sample plate used for mounting solid samples on the SAXS / WAXS beamline When in use, the sample plates are mounted on a vertical post attached to the breadboard of the sample stage with alignment and movement between samples performed with the X & Y motors of the sample stage. The fine precision of the motors on the sample stage allows fine alignment using the view provided by the on-axis camera. A broader view of the sample position may be provided by a flea camera or the Sony camera to check which sample/hole is in the beam and to read sample labels marked directly onto the mounting plate. It is recommended to label a plate of samples so that a wide view of the sample position will help to indicate which sample is in position. Powder samples are usually mounted in capillaries, the mounts for which are detailed below under Solution Samples. They may be mounted by fixing them to a sample plate if requested but this is not recommended due to the fragility of the capillaries. Back to Index. Liquid & Solution SamplesLiquids, gels, solutions and powdered samples are typically run by filling 1.5 mm diameter quartz capillaries mounted using one of the three sample holders available at the beamline. Glass or Special Glass capillaries may also be used to mount samples, but quartz provides a lower background and is recommended for most samples. Samples holders are optimised for 1.5mm capillaries. Capillaries of in the diameter range of 1-2 mm may be accommodated. Capillaries less than 1mm diameter are not suitable and capillaries 2mm or larger may not fit in current sample holders. Many diverse sample types have been successfully mounted at the beamline, including protein samples from 0.1 mg/mL to 10 mg/mL concentrations. If considering weakly scattering samples and/or low concentrations, please refer to the Instrument Background section of the beamline Specifications to check whether a signal will be observed. For liquid and solution samples, a minimum of 20 µL of sample is required and at least 50 µL is preferable. Small volumes or weakly scattering solutions, e.g. proteins, may also be studied using a single, suspended capillary (using a syringe pump to load and eject sample) to provide a consistent background. A syringe pump is used to load and discharge samples without moving the capillary to ensure the most consistent possible background. Output from an FPLC may also be routed directly through the capillary to allow data collection as samples are run on a chromatography column. In all cases sample alignment is similar to that of solid samples with the on-axis camera used to view the sample and the motors of the sample stage used for positioning. Additional cameras may be used to provide wider views of the sample stage as required.
A single 1.5mm quartz capillary mounted on the beamline using a tuohy borst fitting. Sample mounts are also available for capillaries that provide a range of experiment conditions. For static liquids a temperature controlled mount (0 - 80°C) can accommodate up to 40 capillaries (see below) with temperature control via a water bath connected to a brass block which is part of the sample holder. The sample holder may be positioned to allowed capillaries to be vertical or horizontal in the beam. As loading a series of 40 capillaries is time consuming samples should be loading in capillaries prior to arriving for an experiment to ensure beam time is well utilised.
The SAXS/WAXS 40 capillary mount with temperature control from 0-80°C
Top view of mount showing slots for capillaries and brass block. A smaller mount may also be available for rapid heating or temperature control (0-80°C) of a single capillary of a mobile liquid using a syringe pump attached to the capillary. If this smaller mount is expected to be necessary please contact beamline staff prior to the experiment. For additional information on temperature control please refer to the Sample Environment section. Back to Index. Liquid Samples - FPLCFor protein samples a BioLogic DuoFlow FPLC (Fast Performance Liquid Chromatograph) is available at the beamline. When in use the output of the FPLC is directed through a single capillary mounted on the beamline (see above) allowing SAXS data collection concurrent with a chromatography run. UV and conductivity measurements included with the FPLC provide a check that samples are running OK and aid in identifying data of interest.
BioLogic DuoFlow FPLC. (Image from Bio-Rad Laboratories online catalogue) If planning to use the FPLC for an experiment, a number of issues must be given consideration. First and foremost, whether using the FPLC is appropriate. For samples in which the formation of dimers or other aggregates is expected, thus convoluting the SAXS pattern, the FPLC may help in providing a single phase sample. However, for protein complexes the FPLC may disrupt or break the complexes of interest if their binding affinity is low. Additionally, as the FPLC dilutes a sample by a factor of 10 to 15 samples must be available in high concentration (10 mg/mL minimum) and in sufficient volume to fill a 50 µL sample loop. Planning for using the FPLC is also essential with regard to equpment and experiment time. No columns are provided with the FPLC and users must supply their own, while time should be allowed for 30 minutes per sample and 60 minutes for changing columns and buffers. If considering using the FPLC for an experiment please contact beamline staff in advance for advice and to ensure it is both available and ready. Back to Index. Gels & PastesGel and paste samples may either be run using a flat plate sample holder or an adapter plate attached to the heating/cooling block of the multi-capillary stage. Two duplicate plates are available to allow samples to be loaded in one holder while they are run in the other. A total of 96 sample positions are available in each plate, 48 in the inner section of the holder (preferred if temperature control is required) and 48 at the edges also. In either case holes in the sample plate are covered with Kapton tape or film to hold the samples. Samples are loaded manually by covering one side of the holes, loading samples and then covering the other side of the hole to provide a sealed holder. Users should note that this manual sample loading requires considerable time that should be allowed for when designing their experiment. Preferably samples should be loaded in advance, with users arriving prior to the beginning of beam time if necessary.
Back to Index. Multiwell PlatesA sample holder has also been developed to hold standard multiwell plates to allow very high sample throughput. As the x-ray beam passes directly through the plates there is a significant background due to scattering from the plastic of the plates. However, for strongly scattering samples or samples featuring diffraction rings the signal is acceptable. For suitable samples running 96 or 384 well plates on the beamline allows for many hundreds of samples to be run during an experiment. Users are strongly recommended to discuss the suitability of this arrangement for their work with beamline staff well in advance, preferably during proposal submission.
Multiwell plate sample holder mounted on the SAXS beamline ready for use.
Examples of multi-well plates that may be run on the SAXS/WAXS beamline. Sample EnvironmentThere is sufficient space at the sample position to mount a variety of specialised pieces of equipment and sample environments. Additional space can be provided by attaching additional sections of breadboard to extend the sample stage if necessary. Rotation stages, syringe pumps, capillary mounts and temperature-controlled stages (using a water bath or electric strip heaters) are among the equipment available at the beamline. More detailed information and specifications are provided below, and we encourage you to contact beamline staff if you have any questions. Specialised sample environments or stages, e.g. high pressure cells, furnace stages, magnetic stages, are not readily available and must be provided by the user. In such cases we recommend you contact beamline staff to ensure the environment is manageable. Temperature ControlTemperature monitoring and/or control is performed using a Lakeshore Model 340 temperature control unit connected to the local network to provide control inside or outside the experimental hutch. The Lakeshore unit is typically used for temperature monitoring although it can be used for temperature control if electric strip heaters are used in a temperature stage. Two temperature-controlled stages are available for capillary-mounted samples, both of which are designed for capillary-mounted samples as shown above. The larger (35 capillary) stage uses a recirculating water bath for temperature control. The smaller (1 - 10 capillary) stage may be used with either a water bath or electrical strip heaters for temperature control. The smaller stage may also be used for mobile solution samples or pumping with temperature control. For both stages a temperatue range of 0 - 80°C is available. A water bath (Huber Ministat-230) is also available at the beamline and is typically used to provide temperature control for capillary stages and for gel samples. It may, instead, be used for temperature control of other sample equipment not in the beam position, e.g. water baths for sample beakers, maintaining temperatures in sample equipment. If required please contact beamline staff in advance of your experiment. Stages - Rotation, Linear, Syringe PumpsA couple of rotation stages are available at the beamline in addition to a range of manual and driven linear stages. Linear stages may be readily installed at the sample position to provide motion independent of the sample stage if required. Motor-driven stages are integrated into the beamline controls for operation inside or outside the experimental hutch. A single axis rotation stage and a two circle goniometer, detailed below, are also available for use at the sample position. As these stages take longer to install at the sample position, please notify staff of your requirements prior to your experiment. Rotation stages
Syringe pumps are available. These are facilitated using a driven linear stage to attach to the plunger of a fixed syringe and may be driven from inside or outside the hutch. A syringe pump set-up is typically used with a single 1.5 mm capillary that is either mounted individually or in the smaller temperature stage if temperature control is required. A Tuohy-Borst adapter provides a connection from a capillary to tubing and the syringe pump, and allows use of very small sample volumes (minimum 20 µL) by loading a sample directly into the capillary. GasesA gas bottle rack is provided outside the experimental hutch with lines running to an outlet station inside the hutch for gases that may be required for experiments. An oxygen monitor is installed in the hutch to ensure a safe environment if non-toxic gases are in use. Lines are available for nitrogen, helium and inert gases.Other gases, e.g. CO2, and bottles of nitrogen and helium gas are available upon request although beamline staff must be advised prior to an experiment. Please note that the beamline is not capable of handling toxic gases at this time. If you intend to bring bottled gas onsite this must be advised in the Experiment Authorisation form at least two weeks before the experiment. Compressed air lines are also installed in the experimental hutch and are available for use during user experiments. The compressed air supply is maintained at around 600 bar although this can be adjusted if required. Some tubing is available for gas & compressed air lines. However, to ensure compatibility, users are encouraged to bring tubing for any equipment brought on site. Back to Index. Miscellaneous EquipmentThe SAXS / WAXS beamline has a good record of being able to accommodate all equipment and sample mounts brought by users for experiments. Miscellaneous equipment must be included on the Detailed Experiment Plan form submitted prior to an experiment and should be also be discussed in advance with beamline staff. However, mounting and positioning miscellaneous equipment can take considerable time, particularly if these requirements are not clear prior to the experiment. If you plan to bring any additional equipment that needs to be mounted close to the sample, e.g. stirrers, pumps or controllers, or plan to bring equipment for sample mounting, e.g. Linkham stages, custom sample holders, please contact beamline staff well in advance of your experiment. This will allow for a more efficient set up and will minimise any losses of time due to mounting & positioning equipment. Back to Index. |
