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A microfabricated fixed path length silicon sample holder improves background subtraction for cryoSAXS.

ABSTRACT: The application of small-angle X-ray scattering (SAXS) for high-throughput characterization of biological macromolecules in solution is limited by radiation damage. By cryocooling samples, radiation damage and required sample volumes can be reduced by orders of magnitude. However, the challenges of reproducibly creating the identically sized vitrified samples necessary for conventional background subtraction limit the widespread adoption of this method. Fixed path length silicon sample holders for cryoSAXS have been microfabricated to address these challenges. They have low background scattering and X-ray absorption, require only 640?nl of sample, and allow reproducible sample cooling. Data collected in the sample holders from a nominal illuminated sample volume of 2.5?nl are reproducible down to q ? 0.02?Å-1, agree with previous cryoSAXS work and are of sufficient quality for reconstructions that match measured crystal structures. These sample holders thus allow faster, more routine cryoSAXS data collection. Additional development is required to reduce sample fracturing and improve data quality at low q.

SUBMITTER: Hopkins JB

PROVIDER: S-EPMC4453169 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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A microfabricated fixed path length silicon sample holder improves background subtraction for cryoSAXS.

Hopkins Jesse B JB Katz Andrea M AM Meisburger Steve P SP Warkentin Matthew A MA Thorne Robert E RE Pollack Lois L

Journal of applied crystallography 20150130 Pt 1

The application of small-angle X-ray scattering (SAXS) for high-throughput characterization of biological macromolecules in solution is limited by radiation damage. By cryocooling samples, radiation damage and required sample volumes can be reduced by orders of magnitude. However, the challenges of reproducibly creating the identically sized vitrified samples necessary for conventional background subtraction limit the widespread adoption of this method. Fixed path length silicon sample holders f ...[more]

PMID: 26089749

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Project description:Most of the sample holders currently used in macromolecular crystallography offer limited storage density and poor initial crystal-positioning precision upon mounting on a goniometer. This has now become a limiting factor at high-throughput beamlines, where data collection can be performed in a matter of seconds. Furthermore, this lack of precision limits the potential benefits emerging from automated harvesting systems that could provide crystal-position information which would further enhance alignment at beamlines. This situation provided the motivation for the development of a compact and precise sample holder with corresponding pucks, handling tools and robotic transfer protocols. The development process included four main phases: design, prototype manufacture, testing with a robotic sample changer and validation under real conditions on a beamline. Two sample-holder designs are proposed: NewPin and miniSPINE. They share the same robot gripper and allow the storage of 36 sample holders in uni-puck footprint-style pucks, which represents 252 samples in a dry-shipping dewar commonly used in the field. The pucks are identified with human- and machine-readable codes, as well as with radio-frequency identification (RFID) tags. NewPin offers a crystal-repositioning precision of up to 10?µm but requires a specific goniometer socket. The storage density could reach 64 samples using a special puck designed for fully robotic handling. miniSPINE is less precise but uses a goniometer mount compatible with the current SPINE standard. miniSPINE is proposed for the first implementation of the new standard, since it is easier to integrate at beamlines. An upgraded version of the SPINE sample holder with a corresponding puck named SPINEplus is also proposed in order to offer a homogenous and interoperable system. The project involved several European synchrotrons and industrial companies in the fields of consumables and sample-changer robotics. Manual handling of miniSPINE was tested at different institutes using evaluation kits, and pilot beamlines are being equipped with compatible robotics for large-scale evaluation. A companion paper describes a new sample changer FlexED8 (Papp et al., 2017, Acta Cryst., D73, 841-851).

Project description:Background:Ca2+ plays an important role in many physiological processes and an accurate study of these signals is important. In modern fluorescence microscopy, a charge-coupled device (CCD) camera is widely deployed for calcium imaging. The ratiometric method is used for the fluorescence dye Fura-2 and Grynkiewitz's formula (Grynkiewicz et al., 1985) is commonly used to convert fluorescence to free Ca2+ concentration ([Ca2+]). But the need to subtract the background signal can lead to a big error in ratiometric calcium measurements. When the error due to background subtraction occurs, the fluorescence ratio of 340 nm divided by 380 nm lights may be twice as large as the actual value. Under conditions when the excitation intensity is not adjusted to ensure the same throughput of the objective lens for ultraviolet dye illumination, the indicator does not gradually bleach out for channels with a wavelength of 340 nm and 380 nm light, which lead to an additional error in determining the concentration of Ca2+. New method:Here we present a new approach for calculating [Ca2+] from the ratiometric fluorescence of Fura-2 dye imaged by a CCD camera. It is designed to optimize [Ca2+] measurements with photobleaching correction without background subtraction error. A mathematical method is also provided for removing the existing underestimated value of fluorescence at an excitation wavelength of 340 nm and compensating for the bleaching rate for both channels with wavelengths of 340 nm and 380 nm using a power function. Results:In cultured neurons, the calculations of the free Ca2+ concentration during Ca2+ transients estimated by the old and new methods, determine it to the same extent. This comparison was made under conditions without errors through background subtraction. If there is this error, the old method calculates [Ca2+] with a much higher, rather than the actual value. Conclusions:We present a modified Grynkiewitz's formula for calculation [Ca2+] for ratiometric dye, such as Fura-2 imaged by a CCD camera, with photobleaching correction without background subtraction error.

Dataset Information

A microfabricated fixed path length silicon sample holder improves background subtraction for cryoSAXS.

Publications

A microfabricated fixed path length silicon sample holder improves background subtraction for cryoSAXS.

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