Abstract
A wide variety of crystallization solutions are screened to establish conditions that promote the growth of a diffraction-quality crystal. Screening these conditions requires the assessment of many crystallization plates for the presence of crystals. Automated systems for screening and imaging are very expensive. A simple approach to imaging trace fluorescently labeled protein crystals in crystallization plates has been devised, and can be implemented at a cost as low as $50. The proteins β-lactoglobulin B, trypsin and purified concanavalin A (ConA) were trace fluorescently labeled using three different fluorescent probes: Cascade Yellow (CY), Carboxyrhodamine 6G (CR) and Pacific Blue (PB). A crystallization screening plate was set up using β-lactoglobulin B labeled with CR, trypsin labeled with CY, ConA labeled with each probe, and a mixture consisting of 50% PB-labeled ConA and 50% CR-labeled ConA. The wells of these plates were imaged using a commercially available macro-imaging lens attachment for smart devices that have a camera. Several types of macro lens attachments were tested with smartphones and tablets. Images with the highest quality were obtained with an iPhone 6S and an AUKEY Ora 10× macro lens. Depending upon the fluorescent probe employed and its Stokes shift, a light-emitting diode or a laser diode was used for excitation. An emission filter was used for the imaging of protein crystals labeled with CR and crystals with two-color fluorescence. This approach can also be used with microscopy systems commonly used to observe crystallization plates.
Highlights
The structure and function of many proteins are often determined by X-ray crystallography, which requires crystals with high regularity
Automated imaging systems address these difficulties by focusing the detection approach on characteristics that are likely to be present in macromolecules but not small molecules, such as the presence of tryptophan
The excitation source used to screen crystallization plates containing protein labeled with Cascade Yellow or Pacific Blue was a 5 mm lightemitting diode (LED; LED Supply, catalog No L3-0U5TH15-1; Fig. 2)
Summary
The structure and function of many proteins are often determined by X-ray crystallography, which requires crystals with high regularity. Two-photon fluorescence has been used with an excitation wavelength of 515 nm (Madden et al, 2011) This method requires scanning a high-intensity light source through a region of interest, using a high numerical aperture lens to achieve a sufficiently high photon density that fluorescing molecules can absorb two photons, at twice the wavelength of the normal absorption, within 10À15–10À16 s of each other (Xu & Webb, 1997). This requires a high-power light source plus a means of scanning the light through the sample and acquiring the emission signal from each scanned point. We present a simple and low-cost approach to visible fluorescence imaging of TFL crystallization screening plates
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