Identification of protein crystals by excitation of intrinsic protein fluorescence

XtalLight 100C

XtalLight 100C is an accessory unit to be mounted on standard laboratory micro- scopes or many available imaging systems.
XtalLight 100C provides the ability of protein crystal detection by intrinsic fluorescence of the amino acid trypro- phane and privides a second light source for monochromatic light used for specific fluorescence excitation of
flurophores.
XtalLight 100C is available as a complete set-up including microscope and CCD-camera.

 

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  • XtalLight 100C a combined UV and monochromatic light source for fluorescence excitation
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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.
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.

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. 

However part of the green light spectrum overlays with the fluorescence spectrum of carboxyrhodamine, therefore a bandpass filter 525 nm with 20 nm spectral width is used for excitation and a 550 nm longpass filter is used to separate the fluorescence light.

Summarized spectra of carboxyrhodamine emission and excitation and the applyied optical filters.

Carboxyrhodamine stained crystals with a 0.5 to 2 % labeling efficiency exite
with 515 – 535 nm spectral width, exposure time: 1.2 s.

Technical Data

UV light source

Mercury arc lamp with 120 W

  Lamp life time > 2000 h

  Motorised shutter and intensity control

Green light source

Green LED 525 nm with 150 LM at 700 mA

  LED life time 50,000 h

  Motorised intensity control

  optional other wavelenghts are available

Filter 

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/green LED intensity, filter setting and shutter

  Manually

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

  Open interfacing to third party software

Light guides

Light guide for UV light 1.5 mm diameter

  Length 1.5 m

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

Light guide for green 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 100C 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