Identification of protein crystals by excitation of intrinsic protein fluorescence

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. 
  • 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 span > 2000 h

  Motorised shutter and intensity control

Filter

Motorised filter change up to three positions:

  Pos 1: Shortpass 385 nm

  Pos 2: (optional) Shortpass 325 nm

  Pos 3: (optional) Filter of choice

Control

Control of UV light intensity, filter setting and shutter

  Manually

  Software control from PC over ethernet XtalLight 100(C) remote software runs on  Linux Windows MAC

Light guide

Light guide for UV light 1.5 mm core diameter

  Length 1.5 m

  Customized length (optional)

UV light optics

Focusing optics for directing UV light onto the sample

  Focal lenght 20 mm built-in blocking filter

Hardware

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

Imaging package

(optional)

Computer

  Mini PC attached to Monitor

  Monitor 22 inch for full camera image display

  Operation system: Linux

 

Colour CCD camera

Camera for adaptation to a microscope

  1600 x 1200 pixels

 

Imaging SW

  Live display of camera image

  Control of camera settings for UV and coloured light

  Easy acquisition of UV images and combinations

  Storage and retrieval of images in a data base

  Short UV exposure times to protect crystals against damage

Positioning and protection

  Manual Stage for positioning of optics

  Manual Stage for positioning of UV protection shield (optional)

Adaptable Microscopes

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

Suitable plates and sealing films

Crystallization plates with low intrinsic fluorescence (low birefringence) and UV suitable sealing films

 

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