Identification of protein crystals by excitation of intrinsic protein fluorescence
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XtalLight 100

  • XtalLight 100 is an accessory unit to be mounted on standard laboratory microscopes or many available imaging systems.
  • XtalLight 100 provides the ability of protein crystal detection by intrinsic fluorescence of the amino acid tryprophane.     
  • XtalLight 100 is available as a complete set-up including micros- cope and CCD-camera. 

Product Series


A fully automated imaging system designed for SBS-plates. Equipped with a built-in microscope with 2 µm resolution.

Product Series


A compact cuvette based dynamic light scattering instrument for classic DLS-applications.

Product Series

SpectroLight 600 is designed to apply in situ dynamic light scattering (in situ DLS) to standard SBS-plates. In situ DLS is capable to detect nucleation non- invasively long before crystals appear.

  • Applications
  • Physical background
  • Technical data
  • References
  • Downloads

Applications

Below, examples are shown for intrinsic fluorescence imaging in crystallization plates of different types illuminated with white light and UV (blue)

Needle shaped crystals of a ribosome inactivating protein type I as a hanging drop on a siliconized coverslip

Needle shaped crystals of a ribosome inactivating protein Type I. XtalLight was combined with an imaging system equipped with a CCD-video camera resolution: 1360 x 1036 pixel.

Needles of salt crystals 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.
Needles of salt crystals 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.

Needles of salt crystals 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.

Physical background

XtalLight 100 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.

Technical Data

UV light source

Mercury arc lamp with 120 W

  Lamp life time > 2000 h

  Motorised shutter and intensity control

Filters 

Motorised filter change up to three positions:

  Pos 1: Shortpass 385 nm

  Pos 2: Shortpass 325 nm

  Pos 3: optional wavelength

Control

Control of UV light intensity, filter setting and shutter

  Manually

  Software control from PC over ethernet (Windows, Linux, Mac)

  Open interfacing to third party software

Light guide

Light guide for UV light 1.5 mm diameter

  Length 1.5 m

  Customized length....................

Dimensions

Table-top case

  Portable unit

  400 mm x 300 mm x 200 mm (LxWxH)

  Weight: approx. 12 kg

  Power consumption: 90 to 264 V, 200 W

Software

XtalLight 100 remote software runs on Linux, Windows and Mac

  No installation required, runs from removable USB-Source

  Control of light source parameters

 

UV optics

Focussing optics for directing excitation light onto the sample

  Focal length 20 mm with built-in blocking filter

  Focal length ______mm

Stage

  Manual stage

Imaging package 

CCD Camera for adaptation to a microscope or to a stage

  1024x768 pixels colour

  1280x1024 pixels colour

 

Computer

Combined Mini PC and  22 inch Monitor for fullcamera image   display

 Linux

Imaging SW

  Live display of camera image

  Control of camera settings for UV and white light

  Easy acquisition of fluorescence light images and combinations

  Storage and retrieval of images in a data base

  Short UV exposure times to protect crystals against damage

Optional Plexiglas UV- Shielding

   Plexiglas® UV 100, 200 x 300 x 3 mm (w, h, t),   UV – transmission 0.3% (DIN EN 410) 

Adaptable Microscopes Adaptable to several microscopes depending on working distance and set-up  

 

References

Karsten Dierks, Arne Meyer, Dominik Oberthür, Gert Rapp, Howard Einspahr, and Christian Betzel

Efficient UV detection of protein crystals enabled by fluorescence excitation at wavelengths longer than 300 nm

 
 
Arne Meyer, Christian Betzel and Marc Pusey
 
Latest methods of fluorescence-based protein crystal identification