4. Spectrometer by yaofenjin


									4. Spectrometer


  - Thermoelectrically cooled CCD enables to detect all available wavelength simultaneously.
  - Capable to detect spectra of ultra low intensity light.
  - For spectrum measurement of bio/chemiluminescence and fluorescence for:
     - FRET (Fluoresence Resonance Energy Transfer)
     - BRET (Bioluminescece Resonance Energy Transfer)
     - Quality control for luminescent/fluorescent reagents


                                                                   (5) CCD

                                 (3) Grating

        (1) Sample cell

                                                                                   (4) Mirror
                                                (2) Collimator

  (1)     Luminescence emitting from the sample cell is led to 45-degree angled mirror through a slit.
  (2)     A collimator collimates the light into a parallel beam and leads it to a diffraction grating.
  (3)     The grating reveal spectrum of the sample emission.
  (4)     A mirror converges the light onto the surface of CCD.
  (5)     A highly sensitive, cooled CCD camera receives the light and measures the intensity in all
          available wavelengths at a time. Cooling makes it possible for spectrum measurement of very
          low-level light with longer exposure time.

Highly Sensitive Spectrometer

  AB-1850 LumiFluor SC

  Advantages of AE-1850 LumiFluor SC

  Issues for conventional spectrometers
  - Commonly used spectrophotometers for bioluminescence and
    chemiluminescence spectral analysis usually equipped with
    photomultiplier tubes (PMT) as detectors. After the sample
    emission is diffracted into several beams by a prism or a
    grating, a PMT detects the light in each wavelength by se-
    quential scanning.
  - The quantum efficiency of the PMT is low comparing to a
                                                                             Data sample
    CCD, resulting bad S/N ratio.                                             Exposure time: 10 sec.
  - The time for scanning has to be extended to detect very low-              Total measurement time: 10 sec.
    level light. It causes to increase total measurement time, but
    long measurement time causes the following:
       - Increasing background noise.
       - Quickly decaying emission is not measured correctly.

  The LumiFluor SC system uses a high optical            1. Emission/fluorescence detection system

  efficiency spectrograph and a thermoelectrically            Spectral wavelength range
                                                              Spectral resolution
                                                                                          350 nm to 750 nm
                                                                                          1 nm
  cooled CCD detector capable of direct imaging of            Exposure time
                                                                                          0.1 to 3600 seconds
                                                                                          TE cooled CCD
                                                              Capacity                    2 x 106 Photons/sec, nm
  full spectra, including weak spectra that require      2. Spectrograph
  extended exposure times.                                   Grating                      150 lines/mm or 600 lines/mm (user
  Unlike scanning spectrometer instruments that               Blaze                       500 nm
                                                         3. Sample compartment
  required prohibitively long integration times, this        Sample volume               10 μL to 100 μL
                                                             Sample cell                 Quartz cell, micro centrifuge tube
  system achieves high S/.N spectra even for             4. Control system (External PC based)
                                                             Functions                    Instrument control
  small sample volumes.                                                                   Emission/fluorescence data analysis and

                                                         Optional excitation source
                                                             Excitation wavelength        350 nm to 500 nm, filter selectable
                                                             Source                       100 W Xenon lamp

Highly Sensitive Spectrometer

  Sample of Application
  LumiFluor for FRET and BRET
     Fluorescence Resonance Energy Transfer (FRET) and Bioluminescence Resonance Energy
     Transfer (BRET) are powerful new tools for the study of protein-protein and protein-nucleic
     acid. The FRET technique is based upon the transfer of light energy from an excited donor
     fluorophore to an acceptor fluorophore. The distance between the two fluorophores and the
     orientation of the fluorophores strongly influences the efficiency of this energy transfer, and is
     characterized by the emission of light of a specific wavelength from the second fluorophore.
     Fluorophore pairs such as BFP (blue fluorescent protein) and GFP (green fluorescent pro-
     tein), or CFP (cyan fluorescent protein) and YFP (yellow fluorescent protein are typically

     BRET is similar to FRET, but rather than using an external excitation source, the donor
     fluorophore is replaced by a luciferin:luciferase complex. By choosing an appropriate lumi-
     nescence and acceptor fluorophore pair, protein interactions can be measured without the
     risk of damage from the excitation source.

     In order to achieve higher levels of sensitivity we have developed a new thermoelectrically
     cooled charge coupled device camera based spectrometer/spectrograph. Our system can
     simultaneously measure the entire spectrum from 350 to 750 nm with a spectral resolution of
     1 nm using only 10 to 100 μL of sample. An optional external excitation source is available
     for FRET analysis, or the system may be used without external excitation for high sensitivity
     BRET analysis.

                                                                            Processing enzyme

                                                                   Vluc-(KR)-YFP          Vluc-(KR)-YFP+PC1

                                                              Reference: Otsuji T, Okuda-Ashitaka E, Kojima S,
                                                              Akiyama H, Ito S, Ohmiya Y. (2004) Anal Biochem.,
                                                              329(2), 230-7: Monitoring for dynamic biological
                                                              processing by intramolecular bioluminescence
   Fig. Spectra of BRET showing intact molecule BRET          resonance energy transfer system using secreted
   emission before and after cleavage at KR site

     Energy from the luciferin/Vargula luciferase reaction (LH2 and Vluc) is transferred to En-
     hanced Yellow Fluorescent Protein (YFP) when these species are in close proximity.
     Yellow light is emitted. Once the molecule is cleaved at the lysine-arginine cleavage site,
     only blue light is emitted from the luciferin/luciferase reaction.


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