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X-RAY RUNS: Apply for Beamtime

2017  Nov 1 - Dec 21

2018  Feb 7 - Apr 3
2018  Proposal/BTR deadline: 12/1/17

2018  Apr 11 - Jun 4
2018  Proposal/BTR deadline: 2/1/18

Macromolecules typically produce only small crystals; to observe diffraction from them (and determine the molecular structure) we need the intense, highly collimated beam from a synchrotron source. And, the tiny beam has to hit the tiny crystal. The location of the beam can be well determined and doesn't change, but how do we know where the crystal is?

The traditional method for imaging protein crystals at most macromolecular crystallography (MX) beamlines employs an on- or off-axis digital bright-field microscope. Accurate visualization of the crystal is necessary to keep it centered in the X-ray beam as it rotates during an oscillation crystallography experiment. Precise centering becomes more difficult when the visibility of the sample is obstructed by ice or excess cryoprotectant. Several methods have been developed in the past decade to address the issues related to sample visualization on the MX beamline. Among others, raster scanning the sample with an attenuated X-ray beam and UV-excited fluorescence are growing in popularity in beamline use for the visualization of microcrystals (>10 μm). However, both UV and X-ray based methods can damage the sample during imaging due to the ionizing nature of radiation at UV and X-ray wavelengths.

Figure 1: Comparison of bright field (BFM) and visible light excited fluorescence (FM) micrographs of a thermolysin crystal at the A1 station. The crystal is mounted in a nylon loop utilizing NVH immersion oil as the cryoprotectant. Crystals were illuminated with a 5 mW 405 nm CW laser and imaged through an FGL435 long-pass Schott glass filter.

A new method for imaging protein crystals was discovered at MacCHESS and is now implemented on both the A1 and F1 stations here (Figure 1). The method utilizes visible light (405 nm) to excite conjugated double bond and delocalized electron systems natively present within a protein crystal. The magnitude of fluorescence is heavily temperature dependent with about a 10-fold stronger fluorescence at cryogenic temperatures. This novel imaging method can also be used for screening crystallization trays using fluorescence confocal microscopy (Figure 2). Unlike methods relying on non-linear optics, visible light excited fluorescence does not occur with salt crystals and thus provides a mild method for discriminating between salt and protein crystals in screening trays [1].

Figure 2: Fluorescence confocal imaging of protein crystals in a screening tray. Left to right: lysozyme, thaumatin, thermolysin and trypsin. Crystals were illuminated with a 405nm CW laser.


[1] Lukk T, Gillilan RE, Szebenyi DME and Zipfel WR. “A visible-light-excited fluorescence method for imaging protein crystals without added dyes” J. Appl. Cryst. (2016). 49, 234–240



Submitted by: Tiit Lukk and Marian Szebenyi, MacCHESS, Cornell University