- Applications
- Physical background
- Technical data
- References
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XtalLight 100C
XtalLight 100C is a combined UV-light and a green light source (100 nm and 20 nm spectral width respectively). The UV-light source is designed for intrinsic protein fluorescence imaging. The green light is predominantly used for trace fluorescence imaging when the protein was covalently labled with the flurophore carboxyrhodamine.
Applications

A single salt crystal in a NEXTAL QIA1 µplate (QIAGEN, Canada Inc.) Lot No. 2181002077, covered with standard sealing film. XtalLight was combined with an imaging system equipped with a CCD-video camera resolution: 1360 x 1036 pixel.

Lysozyme crystals covalently stained with Atto 550 Protein Labelling Kit prior to crystallisation. The crystals were illuminated with 525 nm for fluorescence imaging

Thaumatin crystal soaked in an Atto 550 Protein Labelling solution and transfered to mother liquer. The crystals were illuminated with 525 nm for fluorescence imaging.

Thaumatin crystals unstained illuminated with a UV-spectrum from 280 to 380 nm and with 525 nm. Intrinsic fluorescence of tryptophan indicated a protein crytal.
Physical background

Intrinsic Fluorescence Imaging
XtalLight 100C uses a filtered mercury arc lamp emission spectrum for intrinsic protein fluorescence excitation

Tryptophan fluorescence excitation is most efficient at 280 nm wavelength. The other aromatic aminoacids, tyrosine, phenylalanine and histidine, could only be excited at shorter wavelengths. Therefore they are of minor importance for in situ intrinsic fluorescence crystal detection (Figure 2).

The opacity of glas coverslips and sealing films reduce the light intensity significantly (Figure 3). Howerver, the characteristics of a filtered mercury arc lamp spectrum compensates the weak opacity and is still sufficient to excite tryptophan fluorescence.

Tryptophan fluorescence is shown in three images of the same glucose isomerase crystal as sitting drop in a 96 Well, CrystalQuick COC plate (greiner bio-one, 609820). All three images were taken using the same exposure time and light sensitivity. On top, the crystal was covered with a quartz cover slip (suprasil). The relative excitation spectrum intensity was calculated for three different proteins (product of the transmission spectrum with the specific molar absorption of the protein) shown on the left side. In the middle the crystal was covered with a standard polymeric film and below with a common glas cover slip. The opacity for wavelengths below 300 nm is significantly reduced when the crystal is covered by glas. However, the appearance of the intrinsic fluorescence seems almost identical, when illuminated with a filtered mercury arc lamp spectrum.

Trace Fluorescence Imaging
Covalently fluorescence labeled proteins with the fluorophore carboxyrhodamine: Absorption and emission maxima: λex 522 nm, λem 550 nm. The colored light source of XtalLight100C provides a green light spectrum (green curve) and the emission maximum and the absorption peak of carboxyrhodamine match exactly.
Technical Data
UV light source |
Mercury arc lamp with 120 W
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Green light source |
Green LED 525 nm with 150 LM at 700 mA
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Filter |
Motorised filter change up to three positions:
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Control |
Control of UV/green LED intensity, filter setting and shutter
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Light guides |
Light guide for UV light 1.5 mm diameter
Light guide for green light 1.5 mm diameter
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Dimensions |
Table-top case
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Software |
XtalLight 100C remote software runs on Linux, Windows and Mac
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UV optics |
Focusing optics for directing excitation light onto the sample
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Stage |
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Imaging package |
CCD Camera for adaptation to a microscope or to a stage
Computer
Imaging SW
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Optional Plexiglas UV- Shielding |
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Adaptable Microscopes | Adaptable to several microscopes depending on working distance and set-up |
Efficient UV detection of protein crystals enabled by fluorescence excitation at wavelengths longer than 300 nm
Karsten Dierks, Arne Meyer, Dominik Oberthür, Gert Rapp, Howard Einspahr and Christian Betzel